Results of human papillomavirus DNA testing with the hybrid capture 2 assay are reproducible

Reproducibility of the Hybrid Capture 2 Test (HC 2) for human papillomavirus (HPV) DNA detection was evaluated by assaying frozen cervical specimens in 1997 and again in 2001 from 1,775 women with normal cervical cytology. Using a cutoff point of 1.0 pg of HPV DNA/ml between a negative and a positive test result, the result of the kappa test for agreement was 0.72 (a kappa value of >0.60 is considered good agreement). Using cutoff points of 1.0 and 10.0 pg/ml between negative and low positive and between low positive and high positive, respectively, the kappa was 0.68 and the linear-weighted kappa was 0.76. The results of this study indicate that HC 2 testing is reproducible even among cytologically normal women with low test values.     

Human papillomavirus genotype specificity of hybrid capture 2

Hybrid Capture 2 (hc2), a clinical test for carcinogenic human papillomavirus (HPV) DNA, has proven to be a sensitive but only modestly specific predictor of cervical precancer and cancer risk. Some of its nonspecificity for clinical end points can be ascribed to cross-reactivity with noncarcinogenic HPV genotypes. However, the reference genotyping tests that have been used for these comparisons are also imperfect. We therefore sought to describe further the HPV genotype specificity of hc2 by comparing the hc2 results to paired results from two related PGMY09/11 L1 primer-based HPV genotyping assays: Linear Array (LA) and its prototype predecessor, the line blot assay (LBA). LA and LBA results were considered separately and combined (detection by either assay or both assays) for 37 individual HPV genotypes and HPV risk group categories (carcinogenic HPV > noncarcinogenic HPV > negative). Baseline specimens from 3,179 of 3,488 (91.5%) women referred to ALTS (a clinical trial to evaluate the management strategies for women with atypical squamous cells of undetermined significance [ASCUS] or low-grade squamous intraepithelial lesions) because of an ASCUS Papanicolaou smear were tested by all three assays. Among single-genotype infections with genotypes targeted by hc2 as detected by either PCR assay, HPV genotype 35 (HPV35) (86.4%), HPV56 (84.2%), and HPV58 (76.9%) were the most likely to test positive by hc2. Among single-genotype infections with genotypes not targeted by hc2 as detected by either assay, HPV82 (80.0%), HPV66 (60.0%) (recently classified as carcinogenic), HPV70 (59.1%), and HPV67 (56.3%) were the most likely to test positive by hc2. Among women who tested negative for carcinogenic HPV by both PCR tests and were positive for noncarcinogenic HPV by either test, 28% of women were hc2 positive. Conversely, 7.8% of all hc2-positive results in this population were due to cross-reactivity of hc2 with untargeted, noncarcinogenic HPV genotypes. In conclusion, hc2 cross-reacts with certain untargeted, noncarcinogenic HPV genotypes that are phylogenetically related to the targeted genotypes, but the degree of cross-reactivity may be less than previously reported.     

Human papillomavirus detection by the hybrid capture II assay with the liquid cell preservation solution Easyfix

The aim of this study is to validate the liquid cell preservation solution Easyfix for DNA detection of the HPV oncogene using the Hybrid Capture II method. 256 specimens were selected for the cytological study, possible biopsy and HPV oncogene search with the Easyfix fixative fluid and the Cervical Sampler transport medium. The results obtained with both mediums are comparable regardless of the cytological type. The relevance of a cytological study combined with the HPV search is stressed. To conclude, it is possible to put forward that the liquid cell preservation solution Easyfix can be used to detect the HPV oncogene using the Hybrid Capture II method.     

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.     

A PCR-based microwell-plate hybrid capture assay for high-risk human papillomavirus

Human papillomavirus (HPV) is associated with cervical cancer. In this study, we developed a high-throughput microwell-plate hybrid capture (MPHC) method for epidemiological studies of high-risk HPV (HRHPV). The results with 1238 cervical specimens from female outpatients showed a concordance rate of 94.3 % between the MPHC and Hybrid Capture II assay. The MPHC assay showed an average HRHPV rate of 29.3 % for high-risk populations in populous cities of China. The established MPHC assay could sensitively and specifically detect 13 types of HRHPV and is suitable for large-scale screening, especially in areas where real-time PCR or fluorescence equipment is unavailable.     

Comparison of the AMPLICOR human papillomavirus test and the hybrid capture 2 assay for detection of high-risk human papillomavirus in women with abnormal PAP smear

Infection with human papillomavirus (HPV) is a necessary step in the progression to cervical cancer. Many methods for HPV testing are currently available, mostly developed to detect pools of HPV types. Hybrid Capture 2 (HC2) is one of the most widely used. A new PCR-based assay, the Roche AMPLICOR HPV test, has been recently developed. Both assays recognize a group of 13 HR HPV types contemporaneously. This study evaluated the performance of both methods for detecting high-grade cervical lesions as a part of management for abnormal PAP smears. The study population was composed of 213 women, all referred to colposcopy and histologic diagnosis following an abnormal PAP test. Biopsy-confirmed high-grade cervical intraepithelial neoplasia was used as a gold standard. Overall agreement was 84.9% with a kappa value of 0.6. When comparing the ability to detect moderate cervical intraepithelial neoplasia (CIN2+) and high-grade cervical intraepithelial neoplasia (CIN3+/cancer), AMPLICOR proved slightly more sensitive than HC2, a finding that is important when HPV testing is used in a triage of borderline smear results. Genotyping of discordant results showed a prevalence of LR-HPV types in HC2 positive/AMPLICOR negative samples, and a similar prevalence of HR- and LR-HPV types in AMPLICOR positive/HC2 negative samples. In conclusion, the study shows that the AMPLICOR assay is more sensitive than HC2, which makes it a valid alternative for routine clinical use.     

Human papillomavirus detection by hybrid capture and its possible clinical use.

AIMS--To determine the sensitivity of the hybrid capture method for human papillomavirus (HPV) detection and potential clinical uses as a screening method for the identification of cervical intraepithelial neoplasia. METHODS--The presence of oncogenic types of HPV was tested for in samples taken from the cervix at colposcopy, and compared with detection by polymerase chain reaction (PCR) in 60 patients. Both sets of results were corrected with the pathology determined by biopsy and smear cytology. RESULTS--Hybrid capture detection showed 86% agreement with PCR. Eighty three percent of CIN 3 lesions, 62% of CIN 2, 59% of CIN 1 and 21% of normal controls were positive for oncogenic HPV types. CONCLUSION--The hybrid capture detection method is reliable, sensitive, and easy to use. The addition of HPV testing to cytological screening would detect a greater proportion of cervical dysplasia with a higher false positive rate.     

Accuracy and interlaboratory reliability of human papillomavirus DNA testing by hybrid capture.

Epidemiologists and clinicians wishing to introduce human papillomavirus (HPV) testing into cervical cancer prevention programs need standardized, reliable, and accurate HPV DNA tests that can detect the full spectrum of pathogenic HPV types. The Hybrid Capture System assay from Digene (hybrid capture assay) is a nonradioactive kit designed to detect 14 HPV types in two groups: a mix of 9 high-risk types associated with anogenital cancer (HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56) and another group of 5 low-risk types associated with condyloma acuminatum (HPV types 6, 11, 42, 43, and 44). The assay yields quantitative data meant to reflect viral concentration. In a study of 199 cervical specimens from women with concurrent Pap smears, we assessed the reliability of the new assay by comparing the hybrid capture assay results from three laboratories. We assessed the accuracy of the hybrid capture assay in comparison with a reference standard of HPV DNA content (multiple testing by several methods in two reference laboratories). We also compared the hybrid capture assay results with the concurrent cytologic diagnoses on the basis of an independent review of each smear by five pathologists. Pairwise interlaboratory agreement rates on HPV positivity for either high-risk or low-risk types ranged from 87 to 94%, and kappa values ranged from 0.61 to 0.83. Among specimens positive for high-risk types (the most important clinical outcome), the interlaboratory correlations of the quantitative data ranged from 0.60 to 0.90.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of a prototype PCR assay and hybrid capture 2 for detection of carcinogenic human papillomavirus DNA in women with equivocal or mildly abnormal papanicolaou smears

We evaluated Hybrid Capture 2 (HC2) and polymerase chain reaction (PCR) results for paired specimens collected at 19,187 visits from 5,026 of 5,060 women participating in the Atypical Squamous Cells of Undetermined Significance/Low-Grade Squamous Intraepithelial Lesion Triage Study (ALTS). We examined the test agreement between HC2 and PCR detection for any of 13 carcinogenic human papillomavirus types targeted by HC2 and compared clinical performance of the 2 tests for detecting concurrent and follow-up cervical intraepithelial neoplasia (CIN) 3 or cancer. The k value for the 2 assays was 0.65 (95% confidence interval, 0.64-0.66), with 82.7% crude agreement. HC2 was more sensitive (93.6% vs 89.3%; P < .0005) but less specific (41.2% vs 48.5%; P < .0005) than PCR for detecting 2-year cumulative CIN 3 or cancer (n = 503). The presence of multiple types as detected by PCR and/or cytologic abnormality increased the likelihood of an HC2+ result. Increased sensitivity of HC2 compared with PCR was surprising, given the theoretical advantages of PCR-based methods for analytic sensitivity. Smaller amounts of material used in PCR could have limited its sensitivity, but our results demonstrate the importance of optimization and standardization of PCR-based assays for clinical applications.     

Detection of HPV DNA in cervical carcinomas by PCR and hybrid capture assay

HPV DNA was detected in exfoliated cervical cells of 73% (85/116) cervical cancer patients by PCR using HPV consensus primers and by hybrid capture assay (HC II) (Digene Corp., USA) in 77 of the 85 cases found HPV positive by PCR. Presence of HPV 16/18 DNA were investigated in the 79 cases by PCR using type specific primers. HPV 16 was detected in 31 (39%) patients, HPV 18 in 7 (8.8%), both HPV 16 and 18 in 19 (24%) and HPVs other than 16/18 in 22 (27.8%) cases. Age and clinical stages had no significant effect on HPV prevalence. Double infection of HPV 16 and 18 was significantly (p<0.05) high in the older patients (56 years or more) compared to younger group. Results indicated that cervical cancers in India are strongly associated with high-risk type HPV infection. HC II assays and PCR results for detection of HPV in cervical smears were comparable.     

Cross-sectional comparison of an automated hybrid capture 2 assay and the consensus GP5+/6+ PCR method in a population-based cervical screening program

In this cross-sectional study, clinical performances of the hybrid capture 2 assay using an automated instrument (i.e., rapid capture system) (hc2-RCS) and the high-risk human papillomavirus GP5+/6+ PCR-enzyme immunoassay (EIA) test were compared using cervical scrape specimens from 8,132 women that participated in a population-based screening trial. The hc2-RCS test scored significantly more samples positive (6.8%) than the GP5+/6+ PCR-EIA (4.8%) (P < 0.0005). This could be attributed largely to a higher positivity rate by the hc2-RCS test for women with cytologically normal, borderline, or mild dyskaryosis. A receiver operator characteristics analysis of the semiquantitative hc2-RCS results in relation to different cytology categories revealed that these differences are owing to differences in assay thresholds. For women classified as having moderate dyskaryosis or worse who also had underlying histologically confirmed cervical intraepithelial neoplasia grade 3 or cervical cancer (> or =CIN3), the hc2-RCS scored 97% (31/32) of samples positive, versus 91% (29/32) by GP5+/6+ PCR-EIA. However, this difference was not significant (P = 0.25). After increasing the hc2-RCS cutoff from 1.0 to 2.0 relative light units/cutoff value of the HPV16 calibrator (RLU/CO), no additional CIN3 lesions were missed by hc2-RCS, but the number of test-positive women with normal, borderline, or mild dyskaryosis was significantly decreased (P < 0.0005). However, at this RLU/CO, the difference in test positivity between hc2-RCS and the GP5+/6+ PCR-EIA was still significant (P = 0.02). The use of an RLU/CO value of 3.0 revealed no significant difference between hc2-RCS and GP5+/6+ PCR-EIA results, and equal numbers of smears classified as > or =CIN3 (i.e., 29/32) were detected by both methods. In summary, both assays perform very well for the detection of >or =CIN3 in a population-based cervical screening setting. However, adjustment of the hc2-RCS threshold to an RLU/CO value of 2.0 or 3.0 seems to produce an improved balance between the clinical sensitivity and specificity for > or =CIN3 in population-based cervical screening.     

Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results.

BACKGROUND AND OBJECTIVES: A persistent infection by high-risk HPV is now considered as the major cause of cervical carcinoma. The use of a single cytological specimen for HPV DNA testing by two different molecular methods was analyzed and validated. STUDY DESIGN: HPV DNA testing by PCR-ELISA and hybrid capture II HPV test (HC-II), was investigated on 317 cytological samples obtained from Italian women. Two hundred twenty-seven women were referred to virological lab for HPV DNA testing during cytological routine screening and 90 during a cytological and virological follow-up after a conization or hysterectomy. RESULTS: Overall, the concordance between the two assays was high (K=0.87). Compared with PCR-ELISA, the HC-II showed a sensitivity of 91.7% and a specificity of 95.4%. Although the analytical sensitivity of the PCR-ELISA was higher, the performance of the two tests did not differ in recognizing HPV DNA positive patients with either low or high-grade squamous intraepithelial lesions (LSIL or HSIL). HPV DNA positivity was directly correlated with the severity of cytological diagnosis (P<0.005). CONCLUSIONS: In view of the comparable results obtained with the two assays and of the ease of use, and higher throughput of HC-II, it seems advisable, with a single cytological specimen, to employ the HC-II test as a first-line assay, either for screening or diagnosis, and to perform reflex PCR on positive samples, if typing of prevalent high risk HPVs is needed.     

Human papillomavirus detection by hybrid capture in paired cervicovaginal lavage and cervical biopsy specimens

Infection of the uterine cervix with human papillomavirus (HPV) is associated with dysplastic lesions that may progress to malignancy. Certain HPV types are associated with higher risk of cervical cancer than other genital HPVs. The goal of this study was to determine if cells obtained by cervicovaginal lavage contain similar HPV types as paired cervical biopsy in women referred because of abnormal cervical cytology. Thirty-four paired lavage and biopsy samples were analyzed for HPV DNA by hybrid capture, using “low risk” (HPV types 6. 11, and related types and “high risk” group (HPV types 16, 18, and related types) HPV. HPV was detected in 24 lavage samples and 18 biopsies. High risk types were predominant. In 14 of 18 HPV-positive biopsies, the paired lavage was also positive for the same HPV group. Four biopsies were HPV-positive at low levels, and the paired lavage was HPV-negative. The mean viral copy numbers of the biopsies from patients with positive and negative lavage samples were 2.7 and 0.1, respectively (P = .02). Ten low level HPV infections were detected by lavage that were not detected by biopsy. HPV detection by hybrid capture in cells obtained by cervicovaginal lavage reflects the results of HPV testing in cervical biopsies. © 1996 Wiley-Liss, Inc.     

Comparison of the Third Wave Invader human papillomavirus (HPV) assay and the digene HPV hybrid capture 2 assay for detection of high-risk HPV DNA

This study compared the clinical performance of the Digene Hybrid Capture 2 (HC2) assay to that of a prototype Third Wave Invader human papillomavirus (HPV) (IHPV) analyte-specific reagent-based assay for the detection of oncogenic or "high-risk" (HR) HPV DNA using liquid-based cytology specimens. In total, 821 ThinPrep vials were tested using both assays. In accordance with the type-specific probes contained within each test, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the IHPV assay were 95.9%, 97.6%, 97.5%, and 96.1%, respectively, and those for the HC2 assay were 98.1%, 86.2%, 87.1%, and 97.9%. Overall, the sensitivity and NPV were comparable between the assays, but the IHPV assay demonstrated a better specificity and PPV, since the IHPV assay had fewer false-positive HR HPV results. The incorporation of an internal control to evaluate the cellularity of the test material is an important feature of the IHPV assay and should reduce the risk of false-negative results due to insufficient sample collection rather than the lack of HR HPV DNA. An additional benefit of the IHPV assay was the smaller sample volume required (1 ml versus 4 ml for the HC2 assay).     

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 of the hybrid capture tube test and PCR for detection of human papillomavirus DNA in cervical specimens.

The strong association of human papillomavirus (HPV) and cervical cancer makes it important to study HPV detection methods that may play a role in cervical cancer screening. We compared two DNA methods that are commonly used for HPV research in the United States: the MY09/MY11 L1 consensus primer PCR-based test and the first-generation Hybrid Capture tube method (HCT). Laboratory assays by each method were performed with 596 cervicovaginal specimens collected from participants in a large cohort study conducted in Portland, Oreg. Included were 499 specimens from women whose cytology was normal and 97 specimens from women with squamous intraepithelial lesions (SILs). The overall HPV DNA positivity for known types was 22.5% by PCR compared to 13.6% by HCT. When the analysis was restricted to the 14 HPV types detectable by both methods, the sensitivity of HCT, with PCR used as the standard for HPV status, was higher for specimens from women with concurrent SILs (81.0%) than for specimens from women with normal cytology (46.7%). Among specimens testing positive by both methods, 97.2% of the time the two methods agreed on whether specimens were positive for cancer-associated HPV types. Both of these HPV test methods provide information that supplements the information provided by the Pap smear. The PCR method has higher analytic sensitivity than HCT in detecting HPV, but HCT may be helpful in identifying women with concurrent SILs.     

Evaluation of the detection of human papillomavirus genotypes in cervical specimens by hybrid capture as screening for precancerous lesions in HIV-positive women

Given the frequency and persistence of human papillomavirus (HPV) infection and associated cytological alterations in HIV-1-positive women, the incidence of uterine cervix neoplasm is likely to increase along with patient survival. More appropriate screening programs, which, in addition to Pap smears (PS), also include tests to detect and type HPV, are needed for the early identification of precancerous cervical lesions. This prospective study involved 168 HIV-positive (group A) and 100 HIV-negative women (group B). Cervicovaginal samples were collected for a PS and HPV DNA search. The detected virus was typed as high–intermediate oncogenic risk HPV (HR-HPV) and low-risk HPV (LR-HPV) using hybrid capture (HC) (Murex-Digene) and in-house PCR tests. The HC-detected prevalence of HPV was 111/168 (66%:HR 75.6%) in group A and 15/100 (15%:HR 42.9%) in group B (P < 0.0001). Polymerase chain reaction (PCR) was positive in 91% and 48%, respectively. No significant difference was observed between drug addicts and heterosexual HIV-1-positive women (P = 0.09). HPV was detected in 94% of the 57 HIV-positive women with cytological alterations. HR-HPV was found in 41/49 women with low-grade and 7/8 with high-grade squamous intraepithelial lesions (LSIL and HSIL, respectively). In women with a negative PS, HPV was detected in 57/111 cases (HR 63%) of group A and in 13/98 of group B (6 cases of HR). Of the 54 group A women who underwent biopsy, histology revealed that 41 had LSIL (18 with negative PS, 19 with LSIL, and 4 with HSIL; HR-HPV in 73% and LR-HPV in 17%), nine had HSIL (5 LSIL and 4 HSIL on cytology; HR-HPV in 89% and LR-HPV in 11%), and four were negative (all cytology negative; 3 HR-HPV and 1 LR-HPV). HR-HPV was more frequent as immunodepression worsened. These results show that cytological evaluation alone underestimated histological alterations in 23/50 women (42.6%), whereas the combination of Pap smear and HPV detection reduced this underestimate to 5%. J. Med. Virol. 56:133–137, 1998. © 1998 Wiley-Liss, Inc.     

Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results

Galan‐Sanchez F, Rodriguez‐Iglesias MA. Use of Cervista HPV HR assay for detection of human papillomavirus in samples with hybrid capture borderline negative results. APMIS 2010; 118: 681–4. We have evaluated Cervista HPV HR for papillomavirus detection in 65 samples previously borderline negative by the hybrid capture method (Digene), using InnoLipa and sequencing as confirmatory techniques. Nine samples were found to be positive by Cervista HPV HR, of which five (7.6%) were confirmed by InnoLipa. Four samples (6.1%) were false positive, of which three samples were reactive for A9 probes. The Cervista HPV HR assay can detect HPV‐positive samples from those with hybrid capture borderline results but can produce false‐positive results when tested for reactivity with A9 probes.     

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.