C ervical cancer is caused by persistent infection with high-risk human papillomavirus (hrHPV). The incidence of invasive cervical cancer has been significantly reduced over the past 50 years by the widespread implementation of cytology-based screening using the Papanicolaou (Pap) test (1). However, because of the limited sensitivity of a single Pap test, cervical cancer and its precursors (cervical intraepithelial neoplasia grades 2 and 3 [CIN2 and CIN3, respectively]) remain significant public health issues. The recent development of assays that allow the detection of hrHPV DNA in cervical specimens has shown them to be more sensitive than the Pap test for the detection of CIN2 and CIN3 in a single screening round (2-4). To be clinically relevant, HPV assays should be based on clinical rather than on analytic sensitivity, since only a minority of HPV-positive women progress to highgrade cervical disease. Moreover, the clinical utility of an HPV test would be enhanced if it could also identify hrHPV-positive women who are at the highest risk of having or developing highgrade disease, because doing so has the potential to improve both sensitivity and specificity. Given the recent evidence for the significantly greater oncogenic risks of HPV16 and HPV18 relative to the other hrHPV types (5-8), an HPV test capable of specifically identifying these 2 genotypes might be expected to provide additional clinical value. Moreover, all HPV tests must demonstrate analytic sensitivity, accuracy, good reproducibility, and inclusivity and exclusivity.The cobas HPV test is a fully automated real-time PCR DNA amplification test that received approval by the Food and Drug Administration in April 2011 and was developed to maximize clinical utility. The cutoff for a positive result was determined based on the ability of the assay to detect histologically confirmed high-grade cervical disease, defined as cervical intraepithelial neoplasia (CIN), grade 2 or worse (ՆCIN2), rather than on the detection of a minimum number of viral copies. The unique test design also allowed simultaneous reporting of a pooled hrHPV result in addition to individual results for HPV16 and HPV18, the 2 most-oncogenic genotypes (5-8). Here, we report details on the development and validation of the cobas HPV test with emphasis on how its design and performance contribute to the establishment of clinical utility. MATERIALS AND METHODSAssay design and conditions. The cobas HPV test is a highly automated assay for the detection of hrHPV DNA in liquid-based cytology (LBC) specimens using real-time PCR technology with a set of 16 PCR primers (8 forward and 8 reverse) that amplify a ϳ200-bp fragment of the L1 gene from all 14 hrHPV genotypes. TaqMan probes labeled with 3 spectrally unique fluorescent dyes allow for the simultaneous detection of 14 hrHPV types from 3 separate channels with real-time PCR technology. Twelve hrHPV types (HPV31, are detected as a pool in channel 1, and the hrHPV16 and HPV18 genotypes are simultaneously detected individually in channel...

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We compared the analytical and clinical performance of cobas® CT/NG for use on the Cobas® 6800/8800 Systems with the Cobas® 4800 CT/NG Test from urogenital and extragenital specimens in over 12,000 specimens from both male and female subjects in Germany and the United States. The analytical sensitivity was ≤40 EB/ml for Chlamydia trachomatis (CT) and ≤1 CFU/ml for Neisseria gonorrhoeae (NG). Using clinical specimens, the overall percent agreement with the Cobas® 4800 CT/NG Test was >98.5%. Across urogenital specimens, there were 93 discrepant specimens; 76 (93.8%) of 81 CT discrepant specimens were 6800+/4800– and 10 (83.3%) of 12 NG discrepant specimens were 6800+/4800–. Sequencing verified CT results for 45 (61.6%) of 73 samples positive by 6800 and 1 (20%) of 5 positive by 4800. Similarly, 7 (70.0%) of 10 NG samples positive by 6800 and 1 of 2 positive by 4800 were confirmed by sequencing. Among discrepant extragenital specimens (all 6800+/4800–), 7 (50%) of 14 oropharyngeal and 23 (76.7%) of 30 anorectal CT discordant samples were confirmed as CT positive by sequencing; all 8 anorectal and 20 (90.9%) of 22 oropharyngeal NG discordant results were also confirmed as NG positive. In conclusion, Cobas® CT/NG for use on the Cobas® 6800/8800 Systems provides high-throughput automated solutions for sexually transmitted infection (STI) screening programs.

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The formation of chemical cross-links between nucleic acids and proteins in formalin-containing media presents challenges for human papillomavirus (HPV) testing of cervical samples collected in SurePath Preservative Fluid. A preanalytic process involving addition of a nucleophilic buffer and heating the sample to 120°C was developed to reverse the effects of cross-linking and improve nucleic acid accessibility for the cobas HPV Test in SurePath. Cycle threshold (C) values for cobas HPV detection were evaluated over time and various temperatures, and mean C differences between pretreated and both untreated SurePath samples and those collected in PreservCyt were assessed. Without pretreatment, low viral levels (1 × limit of detection) of HPV were no longer detectable by 7 days. For prospectively collected specimens, mean (95% CI) C differences between pretreated and untreated samples indicated enhanced HPV DNA recovery in all categories of treated samples: -2.58 (-3.16 to -2.01), -2.63 (-3.62 to -1.64), and -3.39 (-4.95 to -1.82), respectively, for other 12 high-risk HPV types, HPV16, and HPV18. Furthermore, mean (95% CI) C differences of pretreated SurePath samples were comparable to simultaneously collected PreservCyt samples: -0.48 (-0.98 to 0.02) and -0.23 (-0.93 to 0.46), respectively, for HPV16 and HPV18; a borderline significant difference [-0.35 (-0.57 to -0.13)] was observed for other 12 high-risk HPV types. This preanalytic procedure therefore ensures a validated, safe, and accurate method for cobas HPV testing in SurePath.