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.     

Clinical validation of Anyplex™ II HPV HR Detection according to the guidelines for HPV test requirements for cervical cancer screening

BackgroundAnyplex™ II HPV HR Detection (Seegene, Seoul, Korea) is a multiplex real-time PCR using tagging oligonucleotide cleavage and extension (TOCE) technology for simultaneous detection and genotyping of 14 high-risk (HR) HPV types, including HPV16 and HPV18.ObjectivesTo evaluate whether the clinical performance and reproducibility of Anyplex™ II HPV HR Detection meet the international consensus guidelines for HPV test requirements for cervical cancer screening [1].Study designThe clinical performance of Anyplex™ II HPV HR Detection for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was determined relative to that of the reference assay, i.e., HR HPV GP5+/6+-PCR-EIA, by analysis of a total of 879 cervical liquid based cytology (LBC) specimens from a screening population, of which 60 were from women with CIN2+. The intra-laboratory reproducibility and inter-laboratory agreement were determined on 509 LBC samples, of which 172 were positive by the reference assay.ResultsAnyplex™ II HPV HR Detection showed a clinical sensitivity for CIN2+ of 98.3% (59/60; 95% CI: 89.1–99.8) and a clinical specificity for CIN2+ of 93.6% (764/816; 95% CI: 89.8–96.1). The clinical sensitivity and specificity were non-inferior to those of HR HPV GP5+/6+-PCR-EIA (non-inferiority score test: P = 0.005 and P = 0.023, respectively). Both intra-laboratory reproducibility (96.8%; 95% CI: 95.3–98.1; kappa value of 0.93) and inter-laboratory agreement (96.0%; 95% CI: 94.3–97.4; kappa value of 0.91) were high.ConclusionsAnyplex™ II HPV HR Detection performs clinically non-inferior to HR HPV GP5+/6+-PCR-EIA. Anyplex™ II HPV HR Detection complies with international consensus validation metrics for HPV DNA tests for cervical cancer screening [1].     

Evaluation of the clinical performance of OncoPredict HPV® SCR assay within the VALGENT-2 framework

Human papillomavirus (HPV) assays used in cervical cancer screening should be clinically validated according to international criteria. OncoPredict HPV® Screening (SCR) is a partial genotyping multiplex real-time PCR assay targeting E6/E7 genes of 13 high-risk (hr) HPVs. OncoPredict HPV® SCR (index assay) identifies HPV-16 and HPV-18 separately, 11 other hrHPV in aggregate and includes quality controls for sample adequacy, DNA extraction efficiency and PCR inhibition. 1300 VALGENT-2 study samples (from women aged 20–60 attending the Scottish cervical cancer screening program) were tested with the index assay and the GP5+/6+ PCR enzyme immunoassay (standard comparator assay). Non-inferior accuracy detecting cervical intraepithelial neoplasia of grade 2 or worse (CIN2+) of the index versus comparator was verified. Intra- and interlaboratory reproducibility of the index was evaluated by overall concordance and Cohen's kappa, using a sub-population (n = 526). Relative sensitivity and specificity for CIN2+ of the index versus comparator were 1.01 (95% confidence interval [CI]: 0.99–1.03) and 1.02 (95% CI: 1.0–1.04), respectively. Noninferiority p values were all ≤0.05, except for CIN3+ in patients ≥30 years. Excellent intra- and interlaboratory reproducibility was shown with concordance >98% and kappas >0.95. OncoPredict HPV® SCR fulfills the three international validation criteria for hrHPV DNA tests in cervical cancer screening.     

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.     

The BD Onclarity HPV Assay on Samples Collected in SurePath Medium Meets the International Guidelines for Human Papillomavirus Test Requirements for Cervical Screening

This study describes a validation of the BD Onclarity HPV (Onclarity) assay using the international guidelines for HPV test requirements for cervical cancer screening of women 30 years old and older using Danish SurePath screening samples. The clinical specificity (0.90, 95% confidence interval [CI] = 0.88 to 0.91) and sensitivity (0.97, 95% CI = 0.87 to 1.0) of the Onclarity assay were shown to be not inferior to the reference assay (specificity, 0.90 [95% CI = 0.88 to 0.92]; sensitivity, 0.98 [95% CI = 0.91 to 1.0]). The intralaboratory reproducibility of Onclarity was 97%, with a lower confidence bound of 96% (kappa value, 0.93). The interlaboratory agreement was 97%, with a lower confidence bound of 95% (kappa value, 0.92). The BD Onclarity HPV assay fulfills all the international guidelines for a new HPV test to be used in primarily screening. This is the first clinical validation of a new HPV assay using SurePath screening samples, and thus the Onclarity HPV assay is the first HPV assay to hold an international validation for both SurePath and ThinPrep.     

Aptima HPV E6/E7 mRNA test is as sensitive as Hybrid Capture 2 Assay but more specific at detecting cervical precancer and cancer

Detection of human papillomavirus (HPV) E6/E7 oncogene expression may be more predictive of cervical cancer risk than testing for HPV DNA. The Aptima HPV test (Gen-Probe) detects E6/E7 mRNA of 14 oncogenic types. Its clinical performance was compared with that of the Hybrid Capture 2 DNA test (HC2; Qiagen) in women referred for colposcopy and those routinely screened. Aptima was also compared with the PreTect HPV-Proofer E6/E7 mRNA assay (Proofer; Norchip) in the referral population. Cervical specimens collected in PreservCyt (Hologic Inc.) were processed for HPV detection and genotyping with the Linear Array (LA) method (Roche Molecular Diagnostics, Laval, Quebec, Canada). Histology-confirmed high-grade cervical intraepithelial neoplasia (CIN 2) or worse (CIN 2+) served as the disease endpoint. On the basis of 1,418 referral cases (CIN 2+, n = 401), the sensitivity of Aptima was 96.3% (95% confidence interval [CI], 94.4, 98.2), whereas it was 94.3% (95% CI, 92.0, 96.6) for HC2. The specificities were 43.2% (95% CI, 40.2, 46.2) and 38.7% (95% CI, 35.7, 41.7), respectively (P < 0.05). In 1,373 women undergoing routine screening (CIN 2+, n = 7), both Aptima and HC2 showed 100% sensitivity, and the specificities were 88.3% (95% CI, 86.6, 90.0) and 85.3% (95% CI, 83.5, 87.3), respectively (P < 0.05); for women ≥ 30 years of age (n = 845), the specificities were 93.9% (95% CI, 92.3, 95.5) and 92.1% (95% CI, 90.3, 93.9), respectively (P < 0.05). On the basis of 818 referral cases (CIN 2+, n = 235), the sensitivity of Aptima was 94.9% (95% CI, 92.1, 97.7) and that of Proofer was 79.1% (95% CI, 73.9, 84.3), and the specificities were 45.8% (95% CI, 41.8, 49.8) and 75.1% (95% CI, 71.6, 78.6), respectively (P < 0.05). Both Aptima and Proofer showed a higher degree of agreement with LA genotyping than HC2. In conclusion, the Aptima test is as sensitive as HC2 but more specific for detecting CIN 2+ and can serve as a reliable test for both primary cervical cancer screening and the triage of borderline cytological abnormalities.     

Comparison of clinical performance of Abbott RealTime High Risk HPV test with that of hybrid capture 2 assay in a screening setting

Randomized trials have produced sound evidence about the efficacy of screening with human papillomavirus (HPV) DNA tests in reducing cervical cancer incidence and mortality. We evaluated the clinical performance and reproducibility of the Abbott RealTime High Risk (HR) HPV test compared with that of the HR hybrid capture 2 (HC2) assay as assessed by a noninferiority score test. A random sample of 998 cervical specimens (914 specimens of cervical intraepithelial neoplasia less severe than grade 2 [相似文献   

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.     

Validation of ThermoFisher's Papspin for human papillomavirus detection in cervicovaginal specimens using PCR with GP5+/GP6+ primers and the Hybrid Capture II assay

The present study aimed to validate ThermoFisher's (Thermo Fisher Scientific, Runcorn, Cheshire, UK) Papspin (PS) for human papillomavirus (HPV) testing by in-house PCR and by the Hybrid Capture II (HC2) assay and to compare the results with those obtained using Specimen Transport Medium (STM) (Digene Diagnostics, Gaithersburg, MD, USA). Forty-five patients underwent conization for known lesions ranging from atypical squamous cells of undetermined significance (ASC-US) with high-risk HPV (hr-HPV) to high-grade squamous intraepithelial lesion (H-SIL/CIN2+) or adenocarcinoma. Two negative controls were included: one patient with post-menopausal bleeding and another from whom an inflammatory cervical sample was taken without conization. Prior to conization, a gynaecologist collected two cervical samples, fixed in PS or STM, from each patient. All but four cases were tested for panHPV (GP5+/GP6+) and specific hr-HPV subtypes (HPV16, 18, 31,33) by PCR using both media and all were processed for HC2. This study demonstrates that both HPV detection techniques work with PS, showing a specificity of 78.3% for HC2 and 92.8% for PCR compared to 83.8% for HC2 and 92% for PCR using STM. The efficacy of detecting HPV in PS-preserved H-SIL/CIN2+ was very high (96% for PCR using PS and 86% for HC2 using PS), which was in the same range as for PCR using STM, and which was only slightly lower than for HC2 using STM (96% and 89%, respectively). The differences were not statistically significant. It is concluded that ThermoFisher's PS is a valid liquid-based cytology medium for cervical samples, convenient for HPV testing by PCR with GP5+/GP6+ primers and by the HC2 assay.     

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.     

International proficiency study of a consensus L1 PCR assay for the detection and typing of human papillomavirus DNA: evaluation of accuracy and intralaboratory and interlaboratory agreement

The PGMY L1 consensus primer pair combined with the line blot assay allows the detection of 27 genital human papillomavirus (HPV) genotypes. We conducted an intralaboratory and interlaboratory agreement study to assess the accuracy and reproducibility of PCR for HPV DNA detection and typing using the PGMY primers and typing amplicons with the line blot (PGMY-LB) assay. A test panel of 109 samples consisting of 29 HPV-negative (10 buffer controls and 19 genital samples) and 80 HPV-positive samples (60 genital samples and 20 controls with small or large amounts of HPV DNA plasmids) were tested blindly in triplicate by three laboratories. Intralaboratory agreement ranged from 86 to 98% for HPV DNA detection. PGMY-LB assay results for samples with a low copy number of HPV DNA were less reproducible. The rate of intralaboratory agreement excluding negative results for HPV typing ranged from 78 to 96%. Interlaboratory reliability for HPV DNA positivity and HPV typing was very good, with levels of agreement of >95% and kappa values of >0.87. Again, low-copy-number samples were more prone to generating discrepant results. The accuracy varied from 91 to 100% for HPV DNA positivity and from 90 to 100% for HPV typing. HPV testing can thus be accomplished reliably with PCR by using a standardized written protocol and quality-controlled reagents. The use of validated HPV DNA detection and typing assays demonstrating excellent interlaboratory agreement will allow investigators to better compare results between epidemiological studies.     

Comparison of clinical and analytical performance of the Abbott Realtime High Risk HPV test to the performance of hybrid capture 2 in population-based cervical cancer screening

The clinical performance of the Abbott RealTime High Risk HPV (human papillomavirus) test (RealTime) and that of the Hybrid Capture 2 HPV DNA test (hc2) were prospectively compared in the population-based cervical cancer screening setting. In women >30 years old (n = 3,129), the clinical sensitivity of RealTime for detection of cervical intraepithelial neoplasia of grade 2 (CIN2) or worse (38 cases) and its clinical specificity for lesions of less than CIN2 (3,091 controls) were 100% and 93.3%, respectively, and those of hc2 were 97.4% and 91.8%, respectively. A noninferiority score test showed that the clinical specificity (P < 0.0001) and clinical sensitivity (P = 0.011) of RealTime were noninferior to those of hc2 at the recommended thresholds of 98% and 90%. In the total study population (women 20 to 64 years old; n = 4,432; 57 cases, 4,375 controls), the clinical sensitivity and specificity of RealTime were 98.2% and 89.5%, and those of hc2 were 94.7% and 87.7%, respectively. The analytical sensitivity and analytical specificity of RealTime in detecting targeted HPV types evaluated with the largest sample collection to date (4,479 samples) were 94.8% and 99.8%, and those of hc2 were 93.4% and 97.8%, respectively. Excellent analytical agreement between the two assays was obtained (kappa value, 0.84), while the analytical accuracy of RealTime was significantly higher than that of hc2. RealTime demonstrated high intralaboratory reproducibility and interlaboratory agreement with 500 samples retested 61 to 226 days after initial testing in two different laboratories. RealTime can be considered to be a reliable and robust HPV assay clinically comparable to hc2 for the detection of CIN2+ lesions in a population-based cervical cancer screening setting.     

Detection and typing of human papillomavirus by e6 nested multiplex PCR

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.     

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.     

Predominance of High-Risk Human Papillomavirus Genotype 16 and 39 in Women with Premalignant and Malignant Cervical Pathology from Raipur,Chhattisgarh: Clinical Evaluation of Tagging Oligonucleotide Cleavage and Extension Mediated Genotyping Assay

Background: Identification of 14 high-risk human papillomavirus (HR-HPV) is immensely important in elucidating molecular epidemiology, patient monitoring and evidence-based treatment. There is paucity of such data from Chhattisgarh state of Central India. The present study has evaluated tagging oligonucleotide cleavage and extension-mediated Anyplex HR-HPV genotyping assay in identification of 14 HR-HPV genotypes attributable to premalignant and malignant cervical lesion in comparison to GP5+/6+ assay, cytology and colposcopy. Materials and Methods: A total of 185 clinically suspected cases of premalignant and malignant cervical lesion were investigated by HR-HPV genotyping, GP5+/6+, cytology and colposcopy. Results: Genotyping assay showed clinical sensitivity and specificity of 86.5% (confidence interval [CI]: 80.7–91.0) and 100% (CI: 86.3–100) respectively and found noninferior to GP5+/6+ assay (P > 0.05). HR-HPV prevalence was 76.3%, 88.4%, 94.8%, 100% and 100% among cervical intraepithelial neoplasia (CIN) Grade I–III, squamous cell carcinoma and adenocarcinoma cases, respectively. The four most common genotypes detected in CIN I–III were HPV 16 (63.9%), HPV 39 (15.0%), HPV 18 (6.0%) and HPV 33 (5.3%). In cervical cancer (CC) cases, HPV 16 (44.4%), HPV 39 (11.1%), dual infection of HPV 16, 18 (11.1%) and triple infection of HPV 16, 18, 33 (11.1%) were the four most identified genotypic aetiologies. A novel coinfection of HR-HPV 35, 39 were found in two and one cases of CIN I and II. Finding of HPV 39 as the second most prevalent genotype was unusual and underscores the importance of genotyping screening. Conclusion: Anyplex HR-HPV assay is arguably the useful assay for better patient management and can be useful for HR-HPV screening by its unique individual genotype identification of all HR-HPV. Finding of HPV 16, 39, 18, 33 and coinfection of 16,18 and 16, 18, 33 in CIN and CC would help vaccine manufacturer to design specific future HPV polyvalent vaccine preparation to curb down the CC-associated mortality.     

Comparison of Onclarity Human Papillomavirus (HPV) Assay with Hybrid Capture II HPV DNA Assay for Detection of Cervical Intraepithelial Neoplasia Grade 2 and 3 Lesions

Analytical and clinical performance validation is essential before introduction of a new human papillomavirus (HPV) assay into clinical practice. This study compares the new BD Onclarity HPV assay, which detects E6/E7 DNA from 14 high-risk HPV types, to the Hybrid Capture II (HC2) HPV DNA test, to concurrent cytology and histology results, in order to evaluate its performance in detecting high-grade cervical lesions. A population of 567 women, including 325 with ≥ASCUS (where ASCUS stands for atypical cells of undetermined significance) and any HC2 result and 242 with both negative cytology and negative HC2 results, were prospectively enrolled for the study. The overall agreement between Onclarity and HC2 was 94.6% (95% confidence intervals [CI], 92.3% to 96.2%). In this population with a high prevalence of disease, the relative sensitivities (versus adjudicated cervical intraepithelial neoplasia grades 2 and 3 [CIN2+] histology endpoints) of the Onclarity and HC2 tests were 95.2% (95% CI, 90.7% to 97.5%) and 96.9% (95% CI, 92.9% to 98.7%), respectively, and the relative specificities were 50.3% (95% CI, 43.2% to 57.4%) for BD and 40.8% (95% CI, 33.9%, 48.1%) for HC2. These results indicate that the BD Onclarity HPV assay has sensitivity comparable to that of the HC2 assay, with a trend to an increased specificity. Moreover, as Onclarity gives the chance to discriminate between the different genotypes, we calculated the genotype prevalence and the absolute risk of CIN2+: HPV 16 was the most prevalent genotype (19.8%) with an absolute risk of CIN2+ of 77.1%.     

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 and analytical performance of the PapilloCheck HPV-Screening assay using the VALGENT framework

BackgroundThe benefit of HPV testing for cervical cancer screening and disease management has been shown in many recent studies and is part of several new evidence-based guidelines. Assessment of emerging HPV tests in this context is essential, using well-annotated samples, such as those generated via the Validation of Genotyping Tests-HPV (VALGENT) framework.ObjectiveOur aim was to assess the PapilloCheck HPV assay in terms of absolute and relative accuracy for primary cervical cancer screening, using a standard comparator test (GP5+/6 + EIA)already validated in randomised trials.Study designType-specific HPV prevalence was stratified by age and cytology grade and compared with the luminex typing assay incorporating a GP5 + 6+ PCR (GP5+/6+ LMNX Assay). Clinical outcomes were compared with GP5+/6 + EIA.ResultsPrevalence of hrHPV types (high-risk HPV) increased with severity of cytology. The concordance between PapilloCheck and the GP5+/6+ LMNX Assay was excellent when assessed at the qualitative hrHPV presence/absence level also at the type-specific level in the whole population and in women over 30 years of age. Absolute clinical sensitivity and specificity of the PapilloCheck was high and ranged between 95.5% and 98.2% for sensitivity and between 82.7% and 91.6% for specificity, depending on the outcome and population.ConclusionThe sensitivity and specificity of this assay for the outcomes of CIN2+ were similar to those of the standard comparator assay, GP5+/6+ EIA.     

Comparison of Human Papillomavirus Detections in Urine,Vulvar, and Cervical Samples from Women Attending a Colposcopy Clinic

While urine-based sampling for human papillomavirus (HPV) is being explored as a simple and noninvasive approach for cervical cancer screening, data comparing HPV genotyping in urine and those in cellular sampling of the cervix and vulva, and their correlation with rigorously confirmed cervical disease status, are sparse. We performed HPV genotyping on voided-urine and clinician-collected vulvar and cervical samples from 72 women undergoing colposcopy. Although urine-based HPV carcinogenic HPV detection was lower (58.3%) than cervical (73.6%) and vulvar (72.1%) detection (P = 0.05 and 0.07, respectively), the agreement of urine HPV with cervical and vulvar HPV was moderate (kappa = 0.55) and substantial (kappa = 0.62), respectively. Urine-based carcinogenic HPV detection had a clinical sensitivity of 80.8% (95% confidence interval [CI] = 60.7 to 93.5) and a specificity of 53.3% (95% CI = 37.9 to 68.3) for diagnosing cervical intraepithelial neoplasia grades 2/3 (CIN2/3) on histology; 90.0% of CIN3 was positive for urine HPV. The corresponding sensitivity and specificity values for vulvar sampling were 92% (95% CI = 74 to 99) and 40.5% (95% CI = 25.6 to 56.7), and those for cervical sampling were 96.2% (95% CI = 80.4 to 99.9) and 40% (95% CI = 25.7 to 55.7), respectively. HPV16 was the most common carcinogenic genotype detectable in 25% of urine, 33.8% of vulvar, and 31.9% of cervical samples overall, with prevalence increasing with cervical disease grade, regardless of the sampling method. Stronger cervical HPV PCR signal strengths were associated with increased frequency of urine HPV detection. In summary, the relatively lower detection rates but comparable clinical performance of urine-based HPV sampling underscore the need for larger studies to evaluate urine-based sampling for cervical cancer screening, epidemiologic studies, and postvaccination HPV disease surveillance.