Development and Characterization of the cobas Human Papillomavirus Test

Abstract

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...

Quality assurance of human papillomavirus (HPV) testing in the implementation of HPV primary screening in Norway: an inter-laboratory reproducibility study

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