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Rijkaart DC Heideman DA Coupe VM Brink AA Verheijen RH Skomedal H Karlsen F Morland E Snijders PJ Meijer CJ 《Journal of clinical microbiology》2012,50(7):2390-2396
Our aim was to investigate whether high-risk HPV (hrHPV) mRNA detection by PreTect HPV-Proofer can be used to stratify hrHPV DNA-positive women of different cytology classes for risk of high-grade cervical intraepithelial neoplasia or worse (cervical precancer or cancer, i.e., cervical intraepithelial neoplasia grade 2 or higher [≥ CIN2]). A total of 375 women participating in population-based screening, with a GP5+/6+-PCR hrHPV DNA-positive cervical scrape with normal cytology (n = 202), borderline or mild dyskaryosis (BMD) (n = 88), or moderate dyskaryosis or worse (>BMD) (n = 85), were enrolled. Cervical scrapes were additionally subjected to HPV16/18/31/33/45 E6/E7 mRNA analysis by PreTect HPV-Proofer (mRNA test). Referral and follow-up policies were based on cytology, hrHPV DNA, and mRNA testing. The primary study endpoint was the number of ≥CIN2 detected within 3 years of follow-up. The mRNA positivity increased with the severity of cytological abnormality, ranging from 32% (64/202) in hrHPV DNA-positive women with normal cytology to 47% (41/88) in BMD and 68% (58/85) in >BMD groups (P < 0.01). Women with ≥ CIN2 were more likely to test positive by mRNA test (63%) than women without evidence of ≥ CIN2 (32%; P < 0.01). A positive mRNA test result conferred an increased ≥ CIN2 risk in hrHPV DNA-positive women with normal cytology, i.e., 0.55 (95% confidence interval [95% CI], 0.34 to 0.76) in mRNA-positive versus 0.20 (95% CI, 0.07 to 0.33) in mRNA-negative women. In hrHPV DNA-positive women with BMD or >BMD, the result of the mRNA test did not influence the ≥ CIN2 risk. In conclusion, mRNA testing by PreTect HPV-Proofer might be of value to select hrHPV DNA-positive women with normal cytology in need of immediate referral for colposcopy. 相似文献
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Dixon EP King LM Adams MD Grønn P Murphy PG Brown CA Brough GH Skomedal H Malinowski DP Fischer TJ 《Journal of virological methods》2008,154(1-2):220-222
Infection with high-risk human papillomavirus (HPV) is known to be associated directly with the development of cervical cancer. Recent data suggests that the detection of E6/E7 mRNA from high-risk HPV types may serve as a better diagnostic method for detecting the presence of cervical pre-cancer than HPV DNA testing. This report details a commercially available nucleic acid isolation protocol which can be used to isolate reproducibly RNA from residual BD SurePath liquid-based cytology specimens stored for up to 28 days, and have demonstrated the quality and quantity of mRNA is sufficient for detection with the NorChip PreTect HPV-Proofer assay. Of the 242 specimens tested in this study, 236 (97.5%) tested positive for U1A internal control gene expression. HPV type 16, 18, 31, 33 or 45 mRNA was detected in 16/20 (80%) of the analyzed high-grade squamous intraepithelial lesion (HSIL) specimens, with a low frequency of HPV mRNA detected in the normal lesions (3%). The presence of HPV E6 expression in a subset of HPV positive specimens was also detected by real-time RT-PCR. These findings confirm that RNA of sufficient quality can be isolated from residual BD SurePath cervical cytology specimens for use in downstream NASBA and RT-PCR-based assays. 相似文献
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Keegan H Mc Inerney J Pilkington L Grønn P Silva I Karlsen F Bolger N Logan C Furuberg L O'Leary J Martin C 《Journal of virological methods》2009,155(1):61-66
Human papillomavirus (HPV) testing using molecular methods in liquid based cytology (LBC) specimens may be useful as an adjunct to cervical screening by cytology. We compared the positivity rate of the commercially available HPV DNA method hybrid capture 2 (hc2) and the commercially available E6/E7 mRNA method PreTect HPV-Proofer in cytological specimens (n=299). LBC specimens collected (n=299) represented the following cervical cytological disease categories: Normal (n=60), borderline nuclear abnormalities (BNA) (n=34), CIN1 (n=121), CIN2 (n=60), CIN3 (n=24). Overall, 69% (205/299) of the cases were positive by hc2 and 38% (112/299) of the cases were positive by PreTect HPV-Proofer. Concordance rates between the two tests were highest in the high-grade cytology cases (CIN2: 67% and CIN3: 83%) and the normal cytology cases (88%) and lowest in the BNA and CIN1 categories (56% and 52%). HPV DNA viral load analyses were carried out on HPV16 (n=55), HPV18 (n=9) and HPV33 (n=13) samples that were positive by PreTect HPV-Proofer. The sensitivity and specificity of PreTect HPV-Proofer and the hc2 DNA test for the detection of high-grade cytology (i.e. CIN2+) were 71.4% and 75.8% vs 100% and 43.7%, respectively. The relatively low detection rate observed by PreTect HPV-Proofer in the whole range of cytological positive cases, combined with a relatively higher specificity and PPV, suggests that PreTect HPV-Proofer may be more useful than hc2 for triage and in predicting high-grade disease. 相似文献
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Raffaella Ghittoni Rosita Accardi Uzma Hasan Tarik Gheit Bakary Sylla Massimo Tommasino 《Virus genes》2010,40(1):1-13
More than 100 different human papillomavirus (HPV) types have been isolated so far, and they can be sub-grouped in cutaneous
or mucosal according to their ability to infect the skin or the mucosa of the genital or upper-respiratory tracts. A sub-group
of human mucosal HPVs, referred to as high-risk HPV types, is responsible for approximately 5% of all human cancers, which
represents one-third of all the tumours induced by viruses. Epidemiological and biological studies have shown that HPV16 is
the most oncogenic type within the high-risk group. Emerging lines of evidence suggest that, in addition to the high-risk
mucosal HPV types, certain cutaneous HPVs are involved in skin cancer. HPV-associated cancers are intimately linked to HPV
persistence and the accumulation of chromosomal rearrangements. The products of the early genes, E6 and E7, of the high-risk
mucosal HPV types play a key role in both events. Indeed, these proteins have developed a number of strategies to evade host
immuno-surveillance allowing viral persistence, and to alter cell cycle and apoptosis control, facilitating the accumulation
of DNA damage/mutations. Often, the two oncoproteins target the same cellular pathways with different mechanisms, showing
a strong synergism in promoting cellular transformation and neutralizing the immune response. Here, we review most of the
findings on the biological properties and molecular mechanisms of the oncoproteins E6 and E7 from mucosal and cutaneous HPV
types. 相似文献
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Basu P Chandna P Bamezai RN Siddiqi M Saranath D Lear A Ratnam S 《Journal of clinical microbiology》2011,49(10):3537-3544
Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance. 相似文献
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Over the last two decades, since the initial discovery of human papillomavirus (HPV) type 16 and 18 DNAs in cervical cancers by Dr. Harald zur Hausen (winner of the Nobel Prize in Physiology or Medicine, 2008), the HPVs have been well characterised as causative agents for cervical cancer. Viral DNA from a specific group of HPVs can be detected in at least 90% of all cervical cancers and two viral genes, E6 and E7, are invariably expressed in HPV‐positive cervical cancer cells. Their gene products are known to inactivate the major tumour suppressors, p53 and retinoblastoma protein (pRB), respectively. In addition, one function of E6 is to activate telomerase, and E6 and E7 cooperate to effectively immortalise human primary epithelial cells. Though expression of E6 and E7 is itself not sufficient for cancer development, it seems to be either directly or indirectly involved in every stage of multi‐step carcinogenesis. Epidemiological and biological studies suggest the potential efficacy of prophylactic vaccines to prevent genital HPV infection as an anti‐cancer strategy. However, given the widespread nature of HPV infection and unresolved issues about the duration and type specificity of the currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of the viral oncoproteins be elucidated in order to provide the basis for development of new therapeutic strategies against HPV‐associated malignancies. This review highlights novel functions of E6 and E7 as well as the molecular mechanisms of HPV‐induced carcinogenesis. Copyright © 2009 John Wiley & Sons, Ltd. 相似文献
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Novel method for detection, typing, and quantification of human papillomaviruses in clinical samples 总被引:10,自引:0,他引:10
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Hart KW Williams OM Thelwell N Fiander AN Brown T Borysiewicz LK Gelder CM 《Journal of clinical microbiology》2001,39(9):3204-3212
We report the development of a novel detection and typing methodology for human papillomaviruses (HPV) based on real-time PCR with the self-probing fluorescent primers known as Scorpions. This technique is quick, simple, specific, sensitive, and capable of estimating viral load per cell. We report the results of over 100 typing reactions performed on cell lines, biopsies, and cervical cytobrush samples which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. We further report preliminary data suggesting a relationship between viral load per cell and grade of cervical disease. 相似文献
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Chouhy D Gil LB Nocito AL Wojdyla D Ornella L Cittadini J Gardiol D Giri AA 《International journal of molecular medicine》2006,18(5):995-1003
In developing countries, the introduction of human papillomaviruses (HPV) DNA testing as an adjunct to cytological screening programs has been delayed due to the lack of high performance and cost effective diagnostic nucleic acid methods. In this study we report the development and evaluation of the L1HPVPCR, a PCR-based method for the detection and typing of five of the most prevalent high-risk HPV types. The L1HPVPCR assay combines amplification with the MY09/11 HPV consensus primer system, liquid hybridization of the PCR products with no radioactive probes and enzyme immunoassay analysis. The technique is a user-friendly system that allows accurate HPV DNA detection and typing with inexpensive instrumentation that could be performed with not sophisticated reagents in almost any laboratory. Different cutoff points for generic and specific HPV detection were determined using reproducibility analysis and receiver operating characteristic curves to ensure good analytical sensitivity and clinical effectiveness. We used the L1HPVPCR assay to estimate the prevalence of HPV infection in 127 women at risk of cervical cancer from the city of Rosario (Argentina), where no epidemiological data has been previously reported. Further, we explored the clinical utility of the L1HPVPCR assay respect the Pap smear using a combined diagnosis of cytology, histology and colposcopy as gold standard. In conclusion, our results indicate that the assay described here provides a tool for accurate HPV DNA testing and could be applied in regions where no commercial tests are available. 相似文献
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The epidemiology of human papillomaviruses (HPVs) genotype distribution of cutaneous warts in Jordanian patients were studied. A total of 200 samples were collected using skin swabs from patients with warts attending the dermatology clinic at the Jordan University Hospital over the period of June 2010 to October 2010. Another 100 control samples were taken from healthy Jordanian individuals with no current or previous history of warts. DNA extraction and sequencing was carried out using PCR with the FAP primer pair to detect HPV DNA, followed by multiple‐type‐specific (Multiplex) PCR combined with DNA sequencing. The prevalence of HPV among Jordanian patients tested with warts diagnosed clinically was 82% (157/192); of these 45% (87/192) were detected by FAP PCR method, and 37% (70/192) were detected by multiplex PCR method. Sequencing of the FAP positive samples shows that HPV 2 was associated with the highest prevalence (36%), followed by HPV 27 (28%) and HPV 57 (21%). A total of 6% of healthy persons were positive for HPV DNA. In conclusion, this study demonstrates that alpha HPV types (HPV 2, HPV 27, and HPV 57) are associated with the most prevalent cutaneous warts in Jordanian patients J. Med. Virol. 85: 1058–1062, 2013. © 2013 Wiley Periodicals, Inc. 相似文献
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Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses 总被引:8,自引:0,他引:8
Jordens JZ Lanham S Pickett MA Amarasekara S Abeywickrema I Watt PJ 《Journal of virological methods》2000,89(1-2):29-37
A novel method for the detection and typing of human papillomavirus (HPV) was developed using molecular beacon primers. The method is based on the use of HPV-specific primers containing a hairpin loop structure in which fluorescent donor and quencher groups are held in close proximity such that fluorescence is quenched. Amplification of the target sequence results in the opening of the loop and the resulting fluorescence can be detected on a sequence detector system (SDS) 7700 (Applied Biosystems), as used for TaqMan assays. Fluorescent amplicons were identified on the SDS 7700 and then typed by a single hybridisation with specific probes immobilised in lines on a nylon membrane and detected on a fluorescent scanner. This novel beacon primer method compared well with conventional PCR for cervical scrape specimens. The combination of the beacon primer method and reverse line blotting should enable large-scale population studies of HPV infection. 相似文献
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目的 研究人乳头状瘤病毒16型(HPVl6)与TPA(12-O-tetradecanog 1phorbo1-13-acetate)协同作用在scid小鼠体内诱发人胚口腔细胞的恶性转化。方法 制备包装含有HPV 16 E6/E7基因的逆转录病毒,用此病毒感染人胚口腔粘膜,将组织块接种于scid小鼠右侧肩部皮下,共分为4组:实验组为感染病毒的口腔粘膜和TPA,共接种8只小鼠;病毒组为感染病毒的口腔粘膜,共接种6只小鼠;促癌组为正常口腔粘膜和TPA,共接种6只小鼠;对照组为正常口腔粘膜,共接种5只小鼠。于接种第3日起在左侧肩部皮下注射TPA,每周一次。观察16周后处死动物,对瘤组织进行病理诊断,并作聚合酶链反应(PCR)检测HPV 16 E6/E7基因。结果 实验组成瘤率为7/8,其他3组成瘤率皆为0/6、0/6、0/5,,实验组肿瘤组织的病理学检查结果证实为生长活跃的纤维组织细胞瘤,在肿瘤组织中用PCR检测到HPV 16 E6/E7基因。结论 口腔细胞在感染含有HPV 16 E6/E7基因的逆转录病毒后,在TPA作用下可以发生恶性转化。 相似文献
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Sasagawa T Minemoto Y Basha W Yamazaki H Nakamura M Yoshimoto H Sakaike J Inoue M 《Virus research》2000,67(2):127-139
We established a new assay to detect the E6-E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene. 相似文献
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Sonia Perez Castro Amparo Iñarrea Fernández María José Lamas González María Teresa Sarán Diez Ana Cid Lama María Jesús Alvarez Martín Mónica Pato Mosquera Isabel López‐Miragaya Nuria Estévez Julio Torres Piñón María Oña Navarro 《Journal of medical virology》2013,85(6):1063-1068
High‐risk human papillomavirus (HPV) DNA detection provides high sensitivity but low specificity for moderate‐grade cervical intraepithelial neoplasia or worse histological identification. A prospective study evaluated mRNA testing efficacy for predicting this histological diagnosis in case of HPV 16 and/or 18 DNA detection. A total of 165 endocervical samples harboring HPV 16 and/or 18 DNA were tested with NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux, Marcy l´Etoile, France). Women with cytological alterations were referred to colposcopy (n = 111). Moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 25.8% of women presenting atypical squamous cells of undetermined significance or low‐grade squamous intraepithelial lesions and in 89.8% of women with high‐grade squamous intraepithelial lesions. mRNA sensitivity was 81.3% and 84.1%, respectively. Specificity was 52.2%, and 80.0%, respectively. Negative predictive value (NPV) was 88.9% in undetermined or low‐grade squamous lesions. Positive predictive value (PPV) was 97.4% in high‐grade squamous lesions. mRNA reduced colposcopies by 44.3% in undetermined or low‐grade squamous lesions. Direct treatment of mRNA‐positive cases reduced 77.5% of colposcopies in high‐grade squamous lesions. Women without cytological alterations were followed for 18 months (n = 35), and moderate‐grade cervical intraepithelial neoplasia or worse was diagnosed in 34.3%; mRNA sensitivity and specificity were 83.3% and 86.9%, respectively. PPV and NPV were 76.9% and 90.9%, respectively for predicting moderate‐grade cervical intraepithelial neoplasia or worse in 18 months. mRNA reduced the number of visits for follow‐up in 62.2%. In conclusion, NucliSENS‐EasyQ® HPV E6/E7‐mRNA‐assay (Biomerieux) can serve as a triage test in case of HPV 16 and/or 18 DNA detection. J. Med. Virol. 85: 1063–1068, 2013. © 2013 Wiley Periodicals, Inc. 相似文献