Evaluation of a Prototype Real-Time PCR Assay for Carcinogenic Human Papillomavirus (HPV) Detection and Simultaneous HPV Genotype 16 (HPV16) and HPV18 Genotyping

Abstract

Results from a prototype real-time PCR assay that separately detected human papillomavirus genotype 16 (HPV16), HPV18, and 12 other carcinogenic HPV genotypes in aggregate (cobas 4800 HPV test) and results from a PCR assay that detects 37 HPV genotypes individually (Linear Array) were compared using a convenience sample of cervical specimens (n ‫؍‬ 531). The percentage of total agreement between the two assays was 94.7% (95% confidence interval, 92.5 to 96.5%). The Linear Array test was more likely than cobas 4800 HPV test to test positive for the 12 other carcinogenic HPV genotypes among women without evidence of cervical disease (P ‫؍‬ 0.004).Persistent cervical infections by approximately 15 carcinogenic human papillomavirus (HPV) genotypes cause cervical cancer and its immediate precursor lesions (17,22,27). DNA testing for carcinogenic HPV has proven itself to be more sensitive, albeit slightly less specific, for the detection of cervical intraepithelial neoplasia lesion grade 2 (CIN2) and more severe lesions (CIN2ϩ) (1,10,11,15,18,19). Carcinogenic HPV DNA testing of cervical samples has been introduced as an adjunct to cervical cytology into primary cervical cancer screening in the United States (20,26), and the International Agency for Research on Cancer has endorsed its use as a stand-alone option in primary cervical cancer screening (14).The introduction of carcinogenic HPV DNA testing with cervical cytology as a primary screening modality creates a clinical dilemma-the management of carcinogenic HPV DNA-positive, cytology-negative women (4), who remain at a low but significant risk of CIN2ϩ. One solution for identifying women at risk for CIN2ϩ in this subgroup is the separate detection of HPV genotype 16 (HPV16) and HPV18, the most carcinogenic HPV genotypes, referring HPV16-or HPV18-positive women immediately to colposcopy and following up with HPV16-and HPV18-negative women after a year (13,26). While the first generation of clinical HPV tests pooled all carcinogenic HPV genotypes and did not include separate HPV genotyping, at least for HPV16 and HPV18, most of the next generation of clinical tests will offer at least HPV16 and HPV18 genotyping. The utility of full HPV genotyping is less certain (2), as it has been shown that repeatedly testing positive for any carcinogenic HPV is a good proxy for HPV genotypespecific persistence, especially in women aged 30 years and older (7).One of the next-generation tests, the cobas 4800 HPV test, detects a pool of 12 carcinogenic HPV genotypes in aggregate, with concurrent, separate detection of HPV16 and HPV18. We conducted the first evaluation of the cobas 4800 HPV test and compared the results to previously reported results (8) generated by a now well-validated Linear Array HPV genotyping test (5, 6, 9, 12) and to cervical diagnoses.We acquired a convenience sample of 552 anonymized residual PreservCyt (PC; Hologic) specimens, after cytologic interpretation and the histologic diagnosis had been rendered, with human subject research approvals as previously ...

Comparison of the cobas Human Papillomavirus (HPV) Test with the Hybrid Capture 2 and Linear Array HPV DNA Tests

“…After taking a specimen for making a conventional Pap smear, a cervical specimen for HPV testing is taken using a sampling kit composed of a collection brush and specimen transport medium (STM; Qiagen, Gaithersburg, MD) for specimen storage and transportation after collection. STM specimens are sent to a central laboratory for routine HPV testing (1,6).…”

“…The cobas HPV test (cobas; Roche Molecular Systems, Pleasanton, CA) was recently approved by the U.S. Food and Drug Administration (FDA) and identifies HPV16 and HPV18 separately as well as detecting a pool of 11 HR-HPV genotypes (HPV31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68) and also HPV66. The test has been validated in some initial studies (1,13), and we sought to further add to the literature by assessing the interassay agreement between cobas and two other well-validated HPV DNA assays using samples collected in specimen transport medium (STM) (Qiagen, Gaithersburg, MD).…”

Comparison of the Abbott RealTi m e High-Risk Human Papillomavirus (HPV), Roche Cobas HPV, and Hybrid Capture 2 Assays to Direct Sequencing and Genotyping of HPV DNA

“…This assay also has been used as a reference test in many studies to evaluate newly developed HPV detection assays (11,21,(25)(26)(27)32). HPV detection assays utilizing the real-time PCR method have also been developed; these are able to produce results for HPV types 16 and 18 and other HR genotypes at once (5,15). Among them, the Abbott RealTime HR HPV assay was introduced to clinical laboratories, demonstrating performances comparable with those of HC2 (4,16,24,33).…”

Commercially available assays for multiplex detection of alpha human papillomaviruses

“…To the best of our knowledge, there is only one paper in the peer-reviewed literature to date evaluating the prototype of the current cobas 4800 HPV Test [74]. At the time of evaluation, a validated clinical cut-off was not available and thus a conservative cut-off of 45 was used to determine positivity.…”

“…At the time of evaluation, a validated clinical cut-off was not available and thus a conservative cut-off of 45 was used to determine positivity. The cobas 4800 HPV Test prototype was comparatively evaluated with the Linear Array HPV Genotyping Test on 531 convenience anonymized residual PreservCyt cervical samples and the overall agreement between the two assays was 94.7% [74]. However, to our knowledge, data on analytical and clinical validation of the cobas 4800 HPV Test are currently being generated; for example, VU University Medical Center (Amsterdam, The Netherlands) is finishing a comparative evaluation of the clinical performance of the cobas 4800 HPV Test and hc2 on samples originating from a population-based screening cohort using recently described evaluation criteria [19,26], and the first preliminary results of an ATHENA trial (an HPV screening study aimed at determining the effectiveness of HPV testing as part of a cervical cancer screening program, which has already recruited 46,867 women from 61 clinical sites across the USA) have been presented at the 26th Annual International Papillomavirus Conference (Montréal, Canada) in July 2010 [75,76].…”