Comparison of the Novel Human Papillomavirus 4 Auto-capillary Electrophoresis Test with the Hybrid Capture 2 Assay and with the PCR HPV Typing Set Test in the Detection of High-Risk HPV Including HPV 16 and 18 Genotypes in Cervical Specimens

The aim of this study was to compare the novel human papillomavirus (HPV) detection method, the HPV 4 Auto-capillary Electrophoresis (ACE) test with the hybrid capture (HC) 2 assay for the detection of high-risk HPVs. In addition, we compared the HPV 4 ACE test with the polymerase chain reaction HPV Typing Set test for the detection of HPV 16 and HPV 18 genotypes. One hundred ninety-nine cervical swab samples obtained from women with previous abnormal Pap smears were subjected to testing with the three HPV tests. The HPV 4 ACE test and the HC 2 assay showed substantial agreement for detection of high-risk HPVs (85.4%, kappa=0.71). The HPV 4 ACE test also showed substantial agreement with the PCR HPV Typing Set test in the detection of HPV 16 and HP V 18 genotypes (89.9%, kappa=0.65). In correlation with cytologic results, the sensitivities and specificities of the HPV 4 ACE test and HC 2 assay were 92.9% vs. 92.9% and 48.1% vs. 50.8%, respectively, when high-grade squamous intraepithelial lesions were regarded as abnormal cytologies. The novel HPV 4 ACE test is a valuable tool for the detection of high-risk HPVs and for genotyping of HPV 16 and HPV 18.     

Detection of high-risk HPV types by the hybrid capture 2 test

A hybrid capture test that can be used to detect at least 13 high-risk HPV types (referred to collectively as HPV-HR) in cervical scrapes (Hybrid Capture 2 probe set B, HC2-B) was evaluated. The HC2-B test is accurate and highly reproducible and the results obtained show excellent agreement with those obtained by a multiplexed type-specific polymerase chain reaction (mts-PCR). An additional assay to identify a subset of HPV-HR types may improve specificity without compromising sensitivity in HC2-B positive specimens with a test result close to the cut-off value given by 1 pg/ml HPV DNA.     

Real-time duplex PCR for simultaneous HPV 16 and HPV 18 DNA quantitation

HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.     

Comparison of DRY and WET vaginal swabs with cervical specimens in Roche Cobas 4800 HPV and Abbott RealTime High Risk HPV tests

BackgroundSelf-collected vaginal swab samples have been proposed as an alternative specimen collection method for human papillomavirus (HPV) DNA detection.ObjectivesTwo vaginal swabs (a cone-shaped flocked swab (DRY) and a L-shape FLOQSwab with 2 mL eNAT transport medium (WET)) were compared to standard cervical samples for HPV DNA testing. Additionally, they were also compared by using Roche Cobas 4800 HPV (Roche_HPV) and Abbott Real-time High Risk HPV (Abbott_HPV) tests.Study designNinety-six women were prospectively enrolled from the National Cancer Center in Korea between June and August 2015. WET and DRY vaginal swabs and cervical specimens were collected. Roche_HPV and Abbott_HPV tests were performed. The Roche_HPV test on cervical specimens was used as reference.ResultsThe observed agreements (kappa) of Roche_HPV and Abbott_HPV between WET and DRY swabs were 89.6% (0.790, 95% confidence interval (95% CI): 0.667–0.913) and 91.7% (0.833, 95%CI: 0.723–0.943), respectively. No statistical difference was observed between WET and DRY swabs (p > 0.05 for all comparisons). For HPV16/18, the sensitivity/specificity of Roche_HPV on the DRY and WET samples presented 93.8%/96.3% and 87.5%/97.5%, respectively. For other High Risk HPV (hrHPV), the sensitivity/specificity of Roche_HPV on the DRY and WET swabs presented 91.9%/91.5% and 97.3%/98.3, respectively. The sensitivity/specificity of the Abbott_HPV on the DRY and WET swabs were 93.8%/98.8%, 87.5%/98.8% for HPV16/18, and 91.9%/93.2%, 100.0%/93.2% for other hrHPV, respectively.ConclusionsHPV tests performed similarly when using vaginal DRY and WET swab samples. Using DRY and WET swabs to collect vaginal specimens could be an alternative to collecting cervical samples for HPV DNA testing.     

Comparison of the Anyplex II HPV28 assay with the Hybrid Capture 2 assay for the detection of HPV infection

BackgroundHuman papillomavirus (HPV) testing is an important part of cervical cancer screening and management of women with abnormal cytology results. The Hybrid Capture 2 (HC2) has been recommended for use as a reference test.ObjectiveTo evaluate a new real-time PCR assay (Anyplex II HPV28) for detecting high risk (HR) HPV and to compare it to the HC2. In addition, we compared the genotyping results of the Anyplex II HPV28 to those of sequencing analysis.Study designA total of 1114 cervical swab specimens were consecutively obtained from a single healthcare center. We submitted all specimens for HPV detection with Anyplex II HPV28 and HC2, then analyzed the discordant results using multiplex PCR followed by direct sequencing.ResultsHC2 detected 72 (6.5%) cases with HR HPV, while Anyplex II HPV28 identified 138 (12.4%) cases. The overall agreement rate was 91.4% (1018/1114) of cases. Discordant results between these two assays were observed in 96 cases; 15 were positive only by HC2, and 81 were positive only by Anyplex II HPV28. Sequencing analyses performed in 80 cases of discordant results revealed 11 false-positive, and 67 false-negative results using HC2 tests and two false-positive results using Anyplex II HPV28.ConclusionsThe Anyplex II HPV28 assay is analytically more sensitive in the detection of the 13 HR types represented by the HC2 assay and exhibited a higher concordance with comprehensive genotyping based on the sequencing analysis, and it could be used as a laboratory testing method for identifying HPV genotypes.     

Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting

Background

The Abbott RealTime High Risk HPV assay (ART) is an automated multiplex real-time PCR test for detection of DNA from 14 high risk (HR) HPV types in cervical specimens and simultaneous distinction of HPV16 and HPV18 from other HR-HPV.

Objectives

To evaluate the performance of the ART assay in specimens referred for HPV testing to our laboratory (referral population) by comparison with historical data from HC2 and INNO-LiPA as well as histological status, if available.

Study design

412 cervical specimens were collected from women between 18 and 70 years of age: 301 previously tested by HC2 without clinical data and 111 previously tested by HC2 and INNO-LiPA with histological diagnosis of CIN3+.

Results

Our study demonstrated good overall agreement between ART, HC2 and INNO-LiPA. In the group of the CIN3+ specimens HR-HPV was detected by ART in 93.07% (95% CI: 88.12-98.02), while HR-HPV detection rates with HC2 and INNO-LiPA were 91.09% (95% CI: 85.53-96.65) and 95.05% (95% CI: 90.82-99.28), respectively. The typing capability of ART for HPV16, HPV18 and a pool of twelve other HR-HPV types was investigated by comparison with INNO-LiPA demonstrating high overall assay concordance (89.81%; k 0.87).

Conclusions

The Abbott RealTime assay showed similar clinical performance for detection of CIN3+ compared with HC2. The high level of automation and ability to identify HPV16, HPV18 and other HR-HPV make this assay a very attractive option for HR-HPV testing, potentially improving patient management by risk stratification of cytological abnormal populations.     

Evaluation of accuracy of three assays for human papillomavirus detection and typing: Hybrid Capture 2, HPV Consensus kit and Amplicor HPV

BACKGROUND: Detection of high-risk human papillomavirus has proved its usefulness in complement of abnormal cervical scrape result. The Hybrid Capture 2 (HC2, Digene) test has proven its efficiency. We have compared this test with HPV Consensus kit (HPVC, Argène) and Amplicor HPV test (AHPV, RocheDiagnostics) on a panel of 88 samples with low HC2 ratios or discordant results between HC2 and cervical scrape. MATERIAL AND METHODS: Cervical samples were tested in parallel by the three methods using a nested amplification of L1 region as reference. RESULTS: Eighty-six samples were suitable for analysis. Results of HC2 and AHPV tests were closely related. The use of a "generic" probe in the HPVC test was responsible for undetermined results, which were not clinically relevant. CONCLUSION: Despite the low viral load of the samples chosen, the hybridization (HC2) and PCR (AHPV or HPVC) methods gave comparable results, with false positive and false negative results for all tests, but a 75% concordance and a high sensibility to detect HPV infection. However, a complementary study on a larger population with ASCUS diagnosis and biopsy under colposcopy would be necessary to valid these assays for a clinical indication.     

Comparison of the Abbott RealTime High-Risk Human Papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 assays to direct sequencing and genotyping of HPV DNA

Infection with high-risk (HR) human papillomavirus (HPV) genotypes is an important risk factor for cervical cancers. We evaluated the clinical performances of two new real-time PCR assays for detecting HR HPVs compared to that of the Hybrid Capture 2 test (HC2). A total of 356 cervical swab specimens, which had been examined for cervical cytology, were assayed by Abbott RealTime HR and Roche Cobas HPV as well as HC2. Sensitivities and specificities of these assays were determined based on the criteria that concordant results among the three assays were regarded as true-positive or -negative and that the results of genotyping and sequencing were considered true findings when the HPV assays presented discrepant results. The overall concordance rate among the results for the three assays was 82.6%, and RealTime HR and Cobas HPV assays agreed with HC2 in 86.1% and 89.9% of cases, respectively. The two real-time PCR assays agreed with each other for 89.6% of the samples, and the concordance rate between them was equal to or greater than 98.0% for detecting HPV type 16 or 18. HC2 demonstrated a sensitivity of 96.6% with a specificity of 89.1% for detecting HR HPVs, while RealTime HR presented a sensitivity of 78.3% with a specificity of 99.2%. The sensitivity and specificity of Cobas HPV for detecting HR HPVs were 91.7% and 97.0%. The new real-time PCR assays exhibited lower sensitivities for detecting HR HPVs than that of HC2. Nevertheless, the newly introduced assays have an advantage of simultaneously identifying HPV types 16 and 18 from clinical samples.     

HIV合并HPV阳性标本中HPV的型别检测

目的探讨深圳地区人类免疫缺陷病毒（HIV）阳性人群中人乳头瘤病毒（HPV）感染分型情况,为HIV阳性人群中HPV感染的防治提供依据。方法运用荧光PCR方法和反向斑点杂交技术对HPV阳性患者进行HPV分型检测。结果在HIV感染者中。利用反向点杂交分型方法对40例HPV阳性标本进行HPV基因分型。其中单型感染有18例（45．0％）,混合感染有19例（47．5％）,共检出16种HPV型别,其中包括11种高危型（16、18、31、33、35、45、52、56、58、68、73型）和5种低危型（6、11、42、44、54型）。在16种型别中,感染率最高的为16型（25．0％）,其次为52型（17．5％）、58型（15．0％）。结论感染HIV人群中生殖器部位HPV的感染率较高,在HIV阳性人群中检出性病相关的高危HPV16／52／58亚型．对HIV阳性人群中HPV感染的防治有重要指导意义。     

Comparison of the Roche Cobas® 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia

BackgroundThe recently FDA (U.S. food and drug administration) approved Roche Cobas^® 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions.ObjectiveEvaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia.Study designA selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array.ResultsOverall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array.ConclusionThe Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia.     

Candidate human papillomavirus (HPV) type 27b: nucleotide sequence and heterogeneity with HPV 27

More than 100 human papillomavirus (HPV) types have been reported as tumorigenic agents. HPV types 2 and 27 were identified in benign tumors and classified closely according to the sequence homology. An HPV type 2 related sequence was identified previously in an oral papilloma. However, the correct typing and sequencing was impossible at that time. A candidate HPV was cloned by the long-PCR technique from an oral papilloma and characterized by nucleotide sequence. This virus was basically HPV type 27, but there was some heterogeneity in comparison with the prototype. Most differences were nucleotide-sequence variations. Two of four-nucleotide insertions were frame shift, resulting in longer L1 protein than that of HPV 27.     

Testing of a new probe 16/18/45 Hybrid Capture (Digene) in women with high risk HPV infection

BACKGROUND: HPV HR detection test are needed when ASCUS is diagnosed on Pap test. The risk of progression to cervical cancer is dependant on the HPV genotype and three types (HPV 16, 18 and 45) are found in 77.4% of the cervical cancer. Here we have tested a new probe 16/18/45 (Digene) that is able to detect specifically these three types. MATERIAL AND METHODS: Thirty-seven women with a Hybrid Capture 2 High Risk test (Digene) positive were selected to test the new probe 16/18/45. Samples were typed using sequencing reaction after GP5+/GP6+ PCR. Types were given after comparison with the GenBank. Discordant results were controlled with Inno-Lipa HPV genotyping v2 test (Innogenetics). RESULTS: Among the 37 women with HR HPV result, 48.6% were positive with the probe 16/18/45 (18 patients). After genotyping, 12 results were concordant and six discordant (three HPV 31, two HPV 58 et one HPV 59). For the other 19 patients with negative result, 18 are concordant and one discordant (HPV 18). Global concordance for typing between this probe and sequencing was 81% with a kappa test of 0.62 that means a good concordance. Positive predictive value is 66.6% and negative predictive value is 94.7%. CONCLUSION: This study shows a good efficiency of the 16/18/45 probe to detect the genotypes that have the higher risk of progression to cervical cancer. This probe could also allow to follow the epidemiology of HR HPV infection after a large use of HPV vaccines.     

Comparison of HPV genotyping assays and Hybrid Capture 2 for detection of high-risk HPV in cervical specimens

High-risk types of human papillomavirus (HR-HPV) are among the primary causes of cervical cancers. Hybrid Capture 2 (HC-II) (Digene, Gaithersburg, MD), which detects 13 HR-HPVs as a group, is the only HPV assay approved to date by the United States Food and Drug Administration. In Korea, several HPV genotyping assays are commercially available, including HPV RFMP (GeneMatrix Co., Seoul), HPVDNACHIP (Biomedlab Co., Seoul), and MyHPV Chip (Mygene Co., Seoul). We compared the results of these assays with those of HC-II. Among 553 residual samples of liquid-based Pap tests, a total of 435 (78.7%) were available for HPV assays. They were classified into four cytologic categories: normal, atypical squamous cells of undetermined significance (ASCUS), low-grade cervical squamous intraepithelial lesions (LSIL), and high grade SIL or carcinoma (HSIL+). Among these samples, 23.0%, 40.6%, 82.5%, 93.8% were HR-HPV positive by HC-II, respectively; 6.6%, 18.1%, 44.4%, 84.4%, by HPV RFMP, respectively; 5.7%, 24.5%, 54.0%, 84.4%, by HPVDNACHIP, respectively; and 6.6%, 11.6%, 42.9%, 84.4%, by MyHPV, respectively. Compared with HC-II, the concordance rates and kappa values were 70.6% and 0.421 for HPV RFMP; 75.4% and 0.514 for HPVDNACHIP; and 67.8% and 0.367 for MyHPV. The concordance rates and kappa values between genotyping assays were 85.1% and 0.644 for HPV RFMP and HPVDNACHIP; 83.4% and 0.574 for HPV RFMP and MyHPV Chip; and 82.8% and 0.579 for HPVDNACHIP and MyHPV Chip. In conclusion, compared with HC-II test, the genotyping tests showed more than fair concordance but lower sensitivity in the detection of HR-HPVs, limiting their usefulness as HR-HPV screening tools.     

Prevalence of different HPV types and estimation of prognostic risk factors based on the linear array HPV genotyping test

The aim of the study was to evaluate the prevalence and risk factors of HPV in a gynecologic population attending outpatient clinics using two new molecular tests. The Amplicor HPV test and the Linear Array (LA) HPV Genotyping test were used for the detection of HPV DNA in 320 women. Multiple logistic regression was used to identify independent prognostic factors of HPV positivity. The agreement between the two methods in terms of their qualitative results was 89.3% (kappa: 0.63). Based on the LA results, the overall prevalence of HPV DNA was 49.1%, 95% confidence interval (95% CI: 43.5%, 54.7%). The prevalence of high‐risk HPV types was 30.3%. The predominant types were HPV‐6 (24.8%) and HPV‐16 (20.4%). Among women with normal cytology, the prevalence of HPV was much higher in those presenting other findings, such as inflammation, than those without other abnormal findings (49.5% vs. 31.5%). On the basis of multivariate analysis, the risk of HPV infection was higher among women with multiple sexual partners [>3 vs. 1: OR = 3.1, 95% CI: (1.5, 7.2)], Pap smear findings [low/high‐grade lesions vs. negative: OR = 2.8, 95% CI: (1.2, 6.5)], the presence of warts [yes vs. no: OR = 3.0, 95% CI: (1.5, 6.3)] and no history of child birth [no vs. yes: OR = 2.6, 95% CI: (1.0, 6.7)]. Younger age was an additional risk factor for HPV infection with carcinogenic genotypes [OR for 1 year increase = 0.93, 95% CI: (0.89, 0.98)]. J. Med. Virol. 81:2059–2065, 2009. © 2009 Wiley‐Liss, Inc.     

Clinical performance of the CLART human papillomavirus 2 assay compared with the hybrid capture 2 test

Persistent infection by high‐risk human papillomavirus (HR‐HPV) is a cause of cervical cancer. The use of HPV detection in cervical screening programs may improve the ability to identify women at risk of cervical cancer. Therefore, the development of appropriate methods for the detection of HR‐HPV is essential. The aim of this study was to evaluate the clinical performance of the CLART Human Papillomavirus 2 assay (CLART) in comparison with the Hybrid Capture 2 test (HC2), using a clinical cut‐off of cervical intraepithelial neoplasia grade 2 or worse. Discrepant results were analyzed further by the PapilloCheck HPV genotyping system. In the 425 studied women, HR‐HPV positivity rates were similar by both tests (CLART‐13 HR‐HPV: 63.1%; CLART‐17 HR‐HPV: 64.7%; HC2: 64.5%). Agreement between CLART‐13 HR‐HPV (κ = 0.969; concordance level 98.6%), CLART‐17 HR‐HPV (κ = 0.974; concordance level 98.8%), and HC2 were very good. When 13 HR‐HPV types were considered, the two tests showed a clinical sensitivity of 96% (95% CI: 92.6–97.9). The clinical specificity of CLART‐13 HR‐HPV was 73.6% (95% CI: 66.7–79.5) for cervical intraepithelial neoplasia grade 2 or worse, which was comparable to HC2 (71.4%; 95% CI: 64.3–77.5). When all 17 HR‐HPV types were considered, CLART showed a clinical sensitivity of 96.9% (95% CI: 93.8–98.5) and a clinical specificity of 71.9% (95% CI: 64.9–78.0). In conclusion, the CLART assay is efficient, sensitive, reproducible, and has a similar performance to HC2 for cervical intraepithelial neoplasia grade 2 or worse. Furthermore, this assay has the advantage of detecting and genotyping 35 HPV types by a single test, which can provide additional information on the predictive value of infection with HR‐HPV. J. Med. Virol. 83:272–276, 2011. © 2010 Wiley‐Liss, Inc.