Detection of human papillomavirus in cervical intra-epithelial neoplasia, using in situ hybridization and various polymerase chain reaction techniques

One hundred and forty-eight randomly chosen neutral-buffered formaldehyde-fixed cervical biopsies in which cervical intra-epithelial neoplasia (CIN) I–III had been diagnosed were tested for HPV (human papilloma virus) DNA by in situ hybridization (ISH) and polymerase chain reaction (PCR). For ISH, we utilized a biotinylated panprobe and type-specific, genomic probe sets. For PCR, we used the general primers GP5/GP6 and their recently described, elongated version GP5+/GP6+, and included the modification of hot-start PCR. Amplified DNA was detected by gel electrophoresis and slot blot hybridization. The positivity rate of ISH was 59% for all biopsies and 69%, 62% and 46% for CIN I, II and III, respectively. The sensitivity of GP5/GP6 was 74% with cold-start PCR and 78% with hot-start PCR. When GP5+/GP6+ was used, the sensitivity increased to 89% with cold-start PCR and to 95% with hot-start PCR. Based on the most sensitive PCR technique, HPV detection was 93%, 95% and 96% in CIN I, II and III, respectively. The number of HPV types decreased with the severity of the lesion, and HPV 16 was the predominant type. Multiple HPVs were rare and almost all HPV-positive cases could be typed. ISH and slot blot hybridization correlated well regarding HPV typing specificity. Our results confirm that distinct HPV types are present in a high proportion of cases of CIN. The sensitivity of ISH is lower than that of PCR. Furthermore, the modified general primers GP5+/GP6+ give a higher yield than GP5/GP6, while hot-start PCR increases sensitivity even further.     

A novel method of HPV genotyping using Hybrid Capture sample preparation method combined with GP5+/6+ PCR and multiplex detection on Luminex XMAP

A novel DNA detection assay comprising Hybrid Capture sample preparation, GP5+/6+ PCR with modifications and Luminex 100 detection was developed and applied to genotyping of human papillomavirus (HPV) in cervical samples. Target-specific sample preparation was performed using magnetic beads conjugated with Hybrid Capture (HC) antibody. DNA-RNA hybrids were formed between DNA target and RNA probes and captured on HC-beads. DNA on magnetic beads was amplified without elution using consensus GP5+/6+ PCR and then genotyped on Luminex beads using hybridization probes for the 17 high-risk HPV types 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73, 82 and an internal control. This new sequence-specific Hybrid Capture sample preparation is fast, efficient and allows direct HPV genotyping by PCR. Compared to traditional non-sequence-specific sample preparation methods, HC sample preparation demonstrated slightly better detection of multiple HPV infections. The clinical utility of this method was demonstrated on cervical samples positive for HR HPV by the Hybrid Capture 2 (hc2) screening assay.     

The Aptima HPV Assay Fulfills the Cross-Sectional Clinical and Reproducibility Criteria of International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening

The Aptima HPV assay (Hologic Gen-Probe, San Diego, CA) is an FDA-approved assay for detecting human papillomavirus (HPV) E6/E7 mRNA from 14 high-risk HPV types. This study evaluated the clinical performance of the Aptima HPV assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+), relative to the high-risk HPV GP5+/GP6+ PCR, in a cross-sectional clinical equivalence analysis using the noninferiority score test with cervical samples from population-based screening, i.e., 69 cervical scraping samples from women with CIN2+ and 843 from women without evidence of CIN2+. In addition, intralaboratory reproducibility over time and interlaboratory agreement of the Aptima HPV assay results were assessed with another set of 548 cervical samples. The Aptima HPV assay showed a clinical sensitivity for CIN2+ of 94.2% (95% confidence interval [CI], 85.5 to 97.8%) and a clinical specificity for CIN2+ of 94.5% (95% CI, 92.8 to 95.9%); by comparison, these figures were 97.1% (95% CI, 89.1 to 99.3%) (67/69 samples) and 93.6% (95% CI, 91.7 to 95.0%) (785/839 samples), respectively, for GP5+/GP6+ PCR. The clinical sensitivity and specificity of the Aptima HPV assay were noninferior to those of GP5+/GP6+ PCR (P = 0.039 and 0.00016, respectively). In addition, high reproducibility of the Aptima HPV assay, as reflected by the intralaboratory reproducibility over time of 96.0% (95% CI, 94.4 to 97.3%) (526/548 samples; kappa = 0.89) and interlaboratory agreement of 96.7% (95% CI, 95.4 to 98.1%) (531/548 samples; kappa = 0.91), was found. Altogether, these data show that the Aptima HPV assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements for cervical screening. Longitudinal data are needed to ensure that the long-term negative predictive value of this mRNA assay is similar to those of validated HPV DNA tests.     

Clinical Evaluation of a GP5+/6+-Based Luminex Assay Having Full High-Risk Human Papillomavirus Genotyping Capability and an Internal Control

The LMNX genotyping kit HPV GP (LMNX) is based on the clinically validated GP5+/6+ PCR, with a genotyping readout as an alternative for the more established enzyme immunoassay (EIA) detection of 14 targeted high-risk human papillomavirus (HPV) types. LMNX is additionally provided with an internal control probe. Here, we present an analysis of the clinical performance of the LMNX using a sample panel and infrastructure provided by the international VALGENT (Validation of Genotyping Tests) project. This panel consisted of cervical specimens from approximately 1,000 women attending routine screening, “enriched” with 300 women with abnormal cytology. Cases were defined as women classified with cervical intraepithelial neoplasia (CIN) grade 2+ (CIN2+) (n = 102) or CIN3+ (n = 55) within the previous 18 months. Controls were women who had normal cytology results over two subsequent screening rounds at a 3-year interval (n = 746). The GP5+/6+-PCR EIA (EIA) was used as a comparator assay and showed sensitivities of 94.1% and 98.2% for CIN2+ and CIN3+, respectively, with a clinical specificity of 92.4% among women aged ≥30 years. The LMNX demonstrated clinical sensitivities of 96.1% for CIN2+ and of 98.2% for CIN3+ and a clinical specificity of 92.6% for women aged ≥30 years. The LMNX and EIA were in high agreement (Cohen''s kappa = 0.969) for the detection of 14 hrHPVs in aggregate, and no significant difference was observed (McNemar''s P = 0.629). The LMNX internal control detected 0.6% inadequate specimens. Based on our study results, we consider the LMNX, similarly to the EIA, useful for HPV-based cervical cancer screening.     

Clinical Impact of the Analytical Specificity of the Hybrid Capture 2 Test: Data from the New Technologies for Cervical Cancer (NTCC) Study

The Hybrid Capture 2 (HC2) test targets 13 human papillomavirus (HPV) types. Here, cross-reactivity with non-HC2-targeted HPV types is described. We aimed to define the proportion of HC2-positive women who had negative results with HC2-targeted HPV types and estimate its determinants and impact on women''s health management. The New Technologies for Cervical Cancer (NTCC) trial was followed in two predetermined phases. Women in the experimental arm were tested for the presence of HPV DNA by HC2 following a sample collection in PreservCyt (first phase) or Digene specimen transport medium (STM) (second phase). HPV genotyping was performed on DNA samples from HC2-positive women by PCR with GP5⁺/GP6⁺ primers and reverse line blot (RLB) hybridization. Untyped samples were submitted to direct sequencing or restriction fragment length polymorphism. Multivariate logistic regression analysis estimated the adjusted odds ratios (ORs) between the presence of HC2-targeted types and age, viral load, and type of transport medium. Out of 2,920 HC2-positive samples, 2,310 (79.1%) were positive on RLB for HC2-targeted types, 396 were positive (13.6%) for only non-HC2-targeted types (mostly represented by HPV-53, HPV-66, and HPV-70), and in 214 (7.33%) samples, no HPV types were detected. The probability of detecting HC2-targeted types increased with increasing viral load expressed as the relative light unit/positive-control specimen ratio (RLU/PC) (OR for unitary increase of log RLU/PC, 1.35; 95% confidence interval [CI], 1.30 to 1.42) and with STM versus PreservCyt (OR, 1.56; 95% CI, 1.25 to 1.84). If only the samples containing HC2-targeted types tested positive, the positive predictive value (PPV) would have increased from 7.0% (95% CI, 6.1% to 8.0%) to 8.4% (95% CI, 7.3 to 9.6), although 4.9% (95% CI, 2.4% to 8.8%) of cervical intraepithelial neoplasia grade 2⁺ (CIN2⁺) cases would have been missed. In conclusion, STM use and an increased cutoff would reduce the HC2 analytical false-positive rate and increase the positive predictive value for high-grade CIN. The gain in clinical sensitivity by detecting non-HC2-targeted HPV types is limited.     

Clinical and Analytical Performance of the Onclarity HPV Assay Using the VALGENT Framework

As the demand for human papillomavirus (HPV)-related cervical screening increases, emerging HPV tests must be evaluated robustly using well-annotated samples, such as those generated in the Validation of HPV Genotyping Tests (VALGENT) framework. Through VALGENT, we assessed the performance of the BD Onclarity HPV assay, which detects 14 high-risk (HR) types and resolves six individual types and three groups of types. Consecutive samples from a screening population (n = 1,000), enriched with cytologically abnormal samples (n = 300), that had been tested previously with the GP5+/6+ PCR enzyme immunoassay (EIA) and the GP5+/6+ PCR LMNX assay (Diassay) were tested with the Onclarity assay. Type-specific HPV prevalences were analyzed according to age and cytological result. The accuracy of the Onclarity assay for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) and CIN3+ was assessed relative to the GP5+/6+ EIA results by using noninferiority criteria. Overall agreement and type-specific agreement between the Onclarity assay and the GP5+/6+ LMNX assay were assessed. The prevalence of HPV types 16, 18, 31, and 45 increased with the severity of cytological results (P for trend, <0.05). For the detection of CIN2+, the Onclarity assay had a relative sensitivity of 1.02 (95% confidence interval [CI], 0.99 to 1.05; P < 0.001 for noninferiority) and a relative specificity of 0.99 (95% CI, 0.97 to 1.00; P = 0.186 for noninferiority). The kappa for agreement between the Onclarity assay and the GP5+/6+ LMNX assay for HR-HPV was 0.92 (95% CI, 0.89 to 0.94), and values for the six individual types ranged from 0.78 (95% CI, 0.68 to 0.87) for HPV-52 to 0.96 (95% CI, 0.93 to 0.99) for HPV-16. These data suggest that the Onclarity assay offers applications for clinical workstreams while providing genotyping information that may be useful for risk stratification beyond types 16 and 18.     

Comparison between the Hybrid Capture II test and a PCR-based human papillomavirus detection method for diagnosis and posttreatment follow-up of cervical intraepithelial neoplasia

Human papillomavirus (HPV) infection is the major cause of cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN), and HPV testing has therefore been proposed for improved triaging and follow-up of women treated for CIN. We compared two common HPV DNA detection tests (Hybrid Capture II [HCII] and PCR-enzyme immunosorbent assay (EIA) using the primers GP5+/GP6+ followed by HPV typing with reverse dot blot hybridization) for sensitivity and specificity for detection of CIN and of CIN recurrence after treatment. Two hundred and thirty-nine women referred to the Department of Obstetrics and Gynaecology in V?ster?s, Sweden, were enrolled because of atypical Pap smears; 177 of these were later treated for dysplasia by conization or loop diathermy. Samples for HPV DNA testing were taken before and 4 to 6 months after treatment. There was substantial agreement between the HCII and PCR-EIA (kappa, 0.70 before treatment and 0.72 after treatment). The sensitivity for histopathologically confirmed CIN III was 100.0% for PCR-EIA and 95.6% for HCII. For patients with CIN II or worse (CIN II+), the sensitivities were 92.9% (PCR-EIA) and 91.8% (HCII). The specificities for CIN II+ in the pretreatment setting were 30.4% for PCR-EIA and 24.1% for HCII. After treatment, the sensitivities for CIN III in cytology were 100.0% by both methods, and for CIN II+, sensitivities were 80.0% by both methods. The specificities for CIN II+ in the posttreatment setting were 83.5% for PCR and 85.4% for HCII. In conclusion, the sensitivities of both PCR-EIA and HCII are high and almost equal, suggesting that both methods are suitable as tools for detection and posttreatment follow-up of CIN II-III.     

Factors associated with HPV persistence after treatment for high-grade cervical intra-epithelial neoplasia with large loop excision of the transformation zone (LLETZ).

BACKGROUND AND OBJECTIVE: Human Papillomavirus (HPV) persistence after high-grade cervical intra-epithelial neoplasia (CIN) removal may be associated with residual lesions or risk of disease recurrence. Knowledge regarding the factors associated with HPV persistence following CIN treatment is still limited. The main purpose of this longitudinal study was to assess the association between characteristics of the patients and their cervical lesions with high-risk HPV-type persistence, detected by commercially available Hybrid Capture II (HC II), after CIN 2 and 3 treatment with large loop excision of the transformation zone (LLETZ). STUDY DESIGN: For this cohort study, a total of 94 women submitted to LLETZ between March 2001 and September 2002 were included. Only women with at least one follow-up visit at 6 or 12 months and confirmed CIN 2 or 3 in the cone specimen were considered. In each visit women answered to a questionnaire and undertook Pap smear and HC II specimens collection. McNemar's, chi-square and Fisher tests were used for univariate analysis. Generalized Estimating Equations (GEE) were used for multivariate analysis. All calculations were performed within 95% confidence intervals (95% CI). RESULTS: Histological evaluation showed 12 (13%) women with CIN, 2 and 82 (87%) with CIN 3 and conization margins were compromised in 27 (29%) cases. Eighty-seven (92%) women showed positive HC II tests prior to LLETZ. Of women initially HPV negative, none had a positive HC II during follow-up. The proportion of positive HPV tests was reduced from 92% to 20%(P < 0.01) at the first visit and to 22% (P < 0.01) at the second visit after LLETZ. Multivariate analysis showed that smoking and age above 35 years (irrespective of margin status) were strongly associated with positive HPV during follow-up. CONCLUSION: HPV persistence following LLETZ was associated with smoking and with the interaction between age and conization margins.     

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma.

Current human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression. To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67-39.76; P<0.0001) compared with the unknown HPV types (OR, 2.87; 95% CI, 0.89-9.22; P=0.08). HPV 16 was the predominant oncogenic HPV type in CIN 2/3 (51%) and carcinoma (71%) and integrated significantly more frequently in carcinoma than in CIN 2/3 (P=0.004). No significant differences in viral load were observed across the disease categories. Our findings suggest that selected genotyping for the eight oncogenic HPV types might be useful in separating women with a higher risk of CIN progression from those with a minimal risk. We also conclude that the HPV 16 integration status has potential to be a marker for risk assessment of CIN progression.     

Human papillomavirus (HPV) DNA triage of women with atypical squamous cells of undetermined significance with cobas 4800 HPV and Hybrid Capture 2 tests for detection of high-grade lesions of the uterine cervix

The triage of women with high-risk (HR) human papillomavirus (HPV)-positive smears for atypical squamous cells of undetermined significance (ASC-US) to colposcopy is now an integrated option in clinical guidelines. The performance of cobas 4800 HPV and that of Hybrid Capture 2 (HC2) for HR HPV DNA detection in cervical samples in PreservCyt were compared in 396 women referred to colposcopy for ASC-US. Of these, 316 did not have cervical intraepithelial neoplasia (CIN), 47 had CIN1, 29 had CIN2 or CIN3 (CIN2+), and 4 had CIN of unknown grade. HR HPV was detected in 129 (32.6%) and 149 (37.6%) samples with HC2 and cobas 4800 HPV, respectively (P = 0.15). The clinical sensitivities and specificities for detecting CIN2+ were 89.7% (95% confidence interval [CI], 72.8 to 97.2%) and 66.7% (95% CI, 61.7 to 71.3%) with cobas 4800 HPV and 93.1% (95% CI, 77.0 to 99.2%) and 72.2% (95% CI 67.4 to 76.5%) with HC2. The performance of cobas 4800 HPV was similar to that of HC2 for identifying women with ASC-US who would benefit the most from colposcopy.     

A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR

In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes. © 1996 Wiley-Liss, Inc.     

应用基因芯片诊断宫颈石蜡组织样本中人乳头状瘤病毒感染及其临床意义

目的探讨应用基因芯片检测宫颈石蜡组织标本中人乳头状瘤病毒（HPV）感染的可能性及其临床意义。方法收集解放军总医院诊断为宫颈鳞状上皮病变的石蜡组织标本40例,其中宫颈浸润性鳞癌18例,宫颈上皮内瘤变（CIN）Ⅲ12例,CINⅠ4例,CINⅡ6例。从组织中提取DNA后采用基因芯片检测23种常见HPV基因亚型,即PCR扩增后产物在基因芯片上进行杂交。同时选用10例经基因芯片检测16型和18型基因阳性的宫颈鳞癌的石蜡组织切片做原位杂交。基因芯片检测结果与部分原位杂交结果进行比较并分析。结果基因芯片检测的18例宫颈鳞癌HPV高危亚型均为阳性（100％）,其中1例为混合阳性;12例CINⅢ中11例为高危亚型阳性（91．7％）,1例阴性;6例CINⅡ的宫颈病变中高危型5例阳性,低危型1例阳性;4例CINⅠ中有2例低危型阳性、2例阴性;宫颈鳞癌和CINⅢ组与CINⅠ和Ⅱ组比较,差异有统计学意义（U=80．0,P〈0．01）。10例宫颈鳞癌基因芯片HPV16型和18型阳性组织中,原位杂交同型探针6例检测显示阳性。结论HPV基因芯片技术可用于检测多种亚型,特异性强,敏感性高,对HPV感染亚型的鉴别及宫颈癌的预防和治疗具有重要意义。     

Clinical Validation of the HPV-Risk Assay,a Novel Real-Time PCR Assay for Detection of High-Risk Human Papillomavirus DNA by Targeting the E7 Region

The HPV-Risk assay is a novel real-time PCR assay targeting the E7 region of 15 high-risk human papillomavirus (HPV) types (i.e., HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -67, and −68), and provides additional genotype information for HPV16 and HPV18. This study evaluated the clinical performance and reproducibility of the HPV-Risk assay with cervical scraping specimens and its utility with self-collected (cervico)vaginal specimens. The clinical performance of the HPV-Risk assay for cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) with cervical scraping specimens was evaluated by a noninferiority analysis, relative to high-risk HPV GP5+/6+ PCR, following international guidelines for HPV test requirements for cervical cancer screening. The HPV-Risk assay showed clinical sensitivity for CIN2+ of 97.1% (95% confidence interval [CI], 89.1 to 99.3%; 67/69 samples) and a clinical specificity for CIN2+ of 94.3% (95% CI, 92.5 to 95.7%; 777/824 samples). The clinical sensitivity and specificity were noninferior to those of GP5+/6+ PCR (noninferiority score test, P = 0.006 and 0.0003, respectively). Intralaboratory reproducibility over time (99.5% [95% CI, 98.6 to 99.8%]; 544/547 samples, kappa = 0.99) and interlaboratory agreement (99.2% [95% CI, 98.6 to 99.8%]; 527/531 samples, kappa = 0.98) for the HPV-Risk assay with cervical scraping specimens were high. The agreement of the HPV-Risk assay results for self-collected (cervico)vaginal specimens and clinician-obtained cervical scraping specimens was also high, i.e., 95.9% (95% CI, 85.1 to 99.0%; 47/49 samples, kappa = 0.90) for self-collected lavage samples and 91.6% (95% CI, 84.6 to 95.6%; 98/107 samples, kappa = 0.82) for self-collected brush samples. In conclusion, the HPV-Risk assay meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements and can be considered clinically validated for cervical screening purposes. The compatibility of the HPV-Risk assay with self-collected specimens supports its utility for HPV self-sampling.     

GP5+/6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes

In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.     

One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization.

This study describes a nonisotopic polymerase chain reaction-reverse hybridization-based method (PCR-RH) for the one-step detection and genotyping of anogenital human papillomavirus (HPV) in a microwell format. HPV DNA was amplified and labeled by PCR using GP5+/GP6+ primers. Labeled amplicons were hybridized to 20 HPV type-specific capture probes anchored to the surface of plastic microwells and detected by an immunoenzymatic assay. Assay sensitivity was <50 pg labeled amplicon, and no cross-reactivity was observed, as determined by hybridizing serial dilutions of labeled PCR products to either matched or mismatched capture probes. The assay was tested on 66 clinical samples (23 specimens with normal histology, I fibropapilloma, 26 cervical intraepithelial neoplasia grade 1 [CIN1], 9 CIN2, and 7 CIN3) and compared with a method based on restriction fragment length polymorphism (RFLP) of PCR products. PCR-RH and PCR-RFLP performed equally well on clinical samples. The overall HPV detection rate was similar: 65.1% (43/66) for PCR-RH and 57.6% (38/66) for PCR-RFLP. HPV DNA was found in all CIN2 and CIN3 samples by both methods; however, PCR-RH detected more positives among normal biopsy samples and CINI cases. Overall, there was good agreement between the two genotyping methods, but RH yielded fewer cases with undetermined HPV genotype.     

Population-based study of screening test performance indices of three human papillomavirus DNA tests

In order to evaluate three common human papillomavirus (HPV) DNA tests for key performance indices in population-based cervical screening, we sampled 12,527 women aged 32-38 years who attended invitational, population-based screening and followed them for 4 years with comprehensive registry linkages. Three different HPV DNA tests (GP5+/6+ general primer PCR (using either AmpliTaq or AmpliTaq Gold DNA polymerase), Amplicor PCR and Hybrid Capture II were evaluated using baseline samples from women who on follow-up developed cervical intraepithelial neoplasia grade 2 or worse (CINII+) (n = 197) as well as a representative subsample of the women in the cohort (n = 794). The population-based HPV prevalence, sensitivity for future cervical intraepithelial neoplasia grade 2 or worse (CINII+), and absolute risk of CINII+ was 7.1%, 87.1%, and 23.2% for AmpliTaq GP5+/6+ PCR, 11.9%, 88.9%, and 11.0% for AmpliTaq Gold GP5+/6+ PCR, 15.7%, 93.4%, and 9.8% for Amplicor, 10.0%, 92.9%, and 15.3% for Amplicor with raised cut-off, and 7.8%, 79.7%, and 16.9% for Hybrid Capture II. In conclusion, AmpliTaq GP5+/6+ PCR and Amplicor with raised cut-off value have adequate performance indices for primary screening.     

Hybrid capture II, a new sensitive test for human papillomavirus detection. Comparison with hybrid capture I and PCR results in cervical lesions

AIM: To test a new assay for the detection of human papillomavirus (HPV) DNA, hybrid capture II (HC II), compared with the previous commercialized hybrid capture I (HC I) and polymerase chain reaction (PCR) results on cervical scrapes from fresh cone excision biopsy samples. METHODS: The three methods were used on cervical scrapes from 42 fresh cone excision biopsy samples. There were nine metaplastic and inflammatory lesions, five low grade lesions, and 28 high grade lesions. PCR was performed using the general primers GP5+/GP6+. The viral load of high risk HPV DNA was estimated by the ratio of relative light units to positive control values in the samples. RESULTS: The sensitivity of HC I for the detection of high grade lesions was 71.4%, while it was 92.8% for HC II and 96.4% for the PCR. Considering only the absence of detectable cervical in situ neoplasia, the specificity was 88.9% for HC I, 66.7% for HC II, and 66.7% for PCR. With HC II, for a ratio of cervical sample to normal control of > 200, the sensitivity for the detection of high grade lesion was only 34.6% with a specificity of 66.7%. CONCLUSIONS: HPV detection with the HC II assay is more sensitive than the previous HC I and represents a more convenient and easier test than PCR for routine use. Nevertheless the viral load estimated with this test cannot be a reliable predictive indicator of high grade lesions.     

Comparison between prototype hybrid capture 3 and hybrid capture 2 human papillomavirus DNA assays for detection of high-grade cervical intraepithelial neoplasia and cancer

We compared the performance of a prototype version of the Hybrid Capture 3 (HC3) human papillomavirus (HPV) DNA assay to the current generation Hybrid Capture 2 (HC2) assay, both of which target 13 oncogenic HPV types, for the detection of cervical intraepithelial neoplasia grade 3 and cancer (CIN3+) with cervicovaginal lavage specimens collected at enrollment into a 10-year cohort study at Kaiser Permanente (Portland, Oreg.). HC3 results for a risk-stratified sample (n = 4,364) were compared to HC2 results for the entire cohort (n = 20,810) with receiver operating characteristics curves, and the optimal cut points for both tests (relative light units [RLU]/positive control [PC]) for the detection of CIN3+ were determined. Specimens were also tested for HPV16 and HPV18 with separate HC3 type-specific probes. The optimal cut point for detecting CIN3+ was 1.0 RLU/PC for HC2, as previously shown, and was 0.6 RLU/PC for HC3. At the optimal cut points, HC3 and HC2 had similar screening performance characteristics for CIN3+ diagnosed at the enrollment visit. In analyses that included cases CIN3+ at enrollment and those diagnosed during early follow-up, HC3 had nonsignificantly higher sensitivity and equal specificity for the detection of CIN3+ compared to HC2; this increase in sensitivity was primarily the result of increased detection of CIN3+ in women who were 30 years of age or older and were cytologically negative (P = 0.006). We also compared the performance of the hybrid capture tests to MY09/11 L1 consensus primer PCR results (n = 1,247). HC3 was less likely than HC2 to test positive for specimens that tested positive by PCR for any untargeted types (P < 0.001). HC3 was less likely than HC2 to test positive for untargeted PCR-detected single infections with HPV53 (P = 0.001) and HPV66 (P = 0.01). There was good agreement between test positivity by PCR and by single type-specific HC3 probes for HPV16 (kappa = 0.76; 95% confidence interval [CI] = 0.71 to 0.82) and for HPV18 (kappa = 0.73; 95% CI = 0.68 to 0.79). In conclusion, we suggest that HC3 (>/=0.6 RLU/PC) may be slightly more sensitive than and equally specific test as HC2 (>/=1.0 RLU/PC) for the detection of CIN3+ over the duration of typical screening intervals.     

Multiple HPV genotypes in cervical carcinomas: improved DNA detection and typing in archival tissues.

BACKGROUND: Human papillomaviruses (HPV) have been considered to be the necessary and central agents of cervical carcinoma. OBJECTIVE: The aim of this study was to determine the prevalence and genotypes of HPV in archival cervical carcinomas. STUDY DESIGN: The study included 152 paraffin-embedded, formaldehyde-fixed cervical carcinoma specimens. To improve the detection and typing of HPV in archival tissues, we conducted a comprehensive study in which, polymerase chain reaction (PCR)-based methods using E7 type-specific (TS) and L1 modified general primers (MY11/GP6+ and GP5+/GP6+) were employed. RESULTS: Overall HPV prevalence was 98% in the cervical carcinomas. HPV 16 was detected in 66% of the tumors, HPV 18 in 22%, HPV 31 in 13%, HPV 33 in 9%, and HPV 58 in 9%. Notably, multiple HPV types were present in 44 (28.9%) of the 152 cervical carcinomas. The most common co-infections were HPV types 16/18 (12 cases), followed by HPV types 16/31 (7 cases). Additionally, HPV 18 was more frequent in adenocarcinomas and adenosquamous carcinomas (86%) than in squamous cell carcinomas (15.8%) (P = 0.0002). CONCLUSIONS: The combination of L1 general primers and E7 type-specific primers can be of use in detecting HPV DNA in archival tissues. The present study showed a high frequency of multiple HPV infections in cervical carcinomas. Hence, relevant HPV typing information in cervical carcinoma is very important for further HPV vaccine design and application.     

P16 and Ki-67 expression improves the diagnostic accuracy of cervical lesions but not predict persistent high risk human papillomavirus infection with CIN1

Objective: To determine the association between the expression of p16 and Ki-67 and cervical lesions, and to evaluate the role of p16 and Ki-67 as prognostic markers for persistent high risk human papillomavirus (hr-HPV) infection. Methods: Totally 1,154 cases of cervical biopsies were enrolled, 331 cases with negative for dysplasia (NEG), 462 with cervical intraepithelial neoplasia 1 (CIN1), 176 with CIN2, 163 with CIN3 and 22 with cervical squamous cell carcinoma (SCC). Furthermore, 283 women with CIN1 were recruited into 12-month follow-up, and HPV specific gene detection by polymerase chain reaction was used to detect hr-HPV of cervical secretions at 6-month-interval for 12-month follow-up period. 40 women were infected with persistent hr-HPV, 182 with transient infection and 61 unfected with hr-HPV. The expression of p16 and Ki-67 were evaluated by immunohistochemical method. The immunostaining results of p16 and Ki-67 were classified into four categories: negative, 1+, 2+ and 3+. Results: There was significant increase in the expression of p16 (P < 0.001) and Ki-67 (P < 0.001) from NEG to SCC. The expression of Ki-67 (P < 0.001) but not p16 (P = 0.254) significantly increased in CIN2, CIN3. Ratio of p16 (P = 0.215) and Ki-67 (P = 0.495) positivity were not correlated with persistent hr-HPV infection. Conclusion: P16 and Ki-67 can improve the diagnostic accuracy of cervical lesions but can not predict persistent hr-HPV infection with CIN1.