Human papillomavirus (HPV) is the central etiological agent for virtually all cervical cancers, for a substantial proportion of other anogenital tract cancers, and for a smaller proportion of head and neck cancers (3
). Currently, more than 100 different HPV types have been identified, and at least 40 types infect the anogenital epithelium. The risk of cancer is not the same for all HPV types. High-risk HPV types include HPV type 16 (HPV-16) and HPV-18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -68, -73, and -82. Low-risk HPV types include HPV-6, -11, -40, -42, -43, -44, -54, -61, -70, -72, -81, and -CP6108. Potentially high-risk types include HPV-26, -53, and -66 (20
). HPV DNA testing has been used (i) for triage of women with a Papanicolaou (Pap) test finding of atypical squamous cells of undetermined significance (ASC-US), (ii) for monitoring for recurrence of a precancerous cervical lesion or cancer after treatment, and (iii) as a primary screening method for cervical cancer in women 30 years old and older (1
The only FDA-approved HPV assay for clinical testing, the Hybrid Capture II (hc2) assay, distinguishes high-risk HPVs from low-risk HPVs but does not provide individual HPV genotyping information. HPV type-specific assays are likely to have a role in the clinical management of the neoplastic diseases associated with HPV infection. Although 60 to 70% of U.S. women become infected with one or more high-risk genital types of HPV during their lifetime, most infections are quickly resolved and without consequence (2
). Women who remain persistently positive for the same high-risk HPV type for extended periods are at increased risk for progression to cancer (11
). HPV genotyping is needed to differentiate women who are repeatedly positive for the same high-risk HPV type from those who are simply sequentially infected with different high-risk types of HPV. As the use of prophylactic HPV vaccines becomes more widespread, surveillance for population-level effectiveness will become an increasingly important activity that is likely to require the use of an HPV type-specific assay (10
). Estimates of the duration of vaccine-induced protection and the potential for herd immunity, for cross-protection, or for replacement (i.e., an increase in the prevalence of non-vaccine-type cervical lesions despite a decrease in the prevalence of vaccine-type lesions) will require HPV type-specific testing. Further, with increased coverage of HPV vaccines and the development of new vaccines that are likely to target more HPV types, the usefulness and methods of HPV testing and genotyping in cervical cancer prevention programs will likely need reevaluation and revision (14
). Currently, HPV genotyping is indispensable for epidemiological and clinical studies of the transmission, natural history, and pathogenesis of HPV, and it is likely to have a role in the management of HPV-related precancerous lesions and cancers in the future.
DNA sequencing is the “gold standard” for HPV genotyping; however, it is costly, time-consuming, and difficult to apply to clinical samples, which frequently have multiple infections and produce nonspecific PCR products. Currently, the most widely used multiplex HPV genotyping assays are reverse line blot (RLB) assays (7
). These assays are based on solid-phase hybridization of amplified HPV sequences to a slot blot membrane. However, RLB assays are labor-intensive, are not easily automated, have limited reproducibility (because they rely on a subjective visual readout) (5
), and are increasingly expensive.
Recent reports support the potential use of the liquid bead microarray (LBMA) assay based on Luminex technology for HPV genotyping, using either the existing GP5+-GP6+ system or the PGMY PCR system (13
). This assay format is sensitive and amenable to high-throughput configuration and potentially can be automated. However, little is known about the analytical sensitivity and specificity of this new assay or about how it performs on clinical specimens in comparison to the other HPV genotyping assays.
In our present study, we developed an LBMA assay based on the MY09-MY11-HMB01 PCR system for genotyping clinically important HPV types (11
). We determined the analytical sensitivity and specificity of the LBMA assay using individual HPV plasmids, compared the genotyping results of the LBMA assay with those of the RLB assay, and estimated the clinical performance of the LBMA assay in comparison to that of the hc2 test using archived cervical swab samples.