Confirmatory Real-Time PCR Assay for Human Papillomavirus (HPV) Type 52 Infection in Anogenital Specimens Screened for HPV Infection with the Linear Array HPV Genotyping Test

The detection of high-risk human papillomavirus (HPV) types in genital specimens has now been approved in several countries for the triage of women with a cytological diagnosis of atypical squamous cells of undetermined significance (ASCUS) and for the primary screening for cervical cancer for women aged 30 years and above as an adjunct to cytology (18). HPV DNA genotyping has proven useful in the study of the natural history and transmission of HPV infection (9, 15). Genotyping assays will be instrumental in assessing the impact of HPV vaccination on the risk of acquisition and on the distribution of individual HPV types in a population. Moreover, HPV genotyping could identify HPVinfected women at greater risk for high-grade cervical intraepithelial neoplasia or cancer (2, 13, 15). The Linear Array HPV genotyping test (LA-HPV) from Roche Molecular Systems is approved by Health Canada for HPV genotyping. LA-HPV identifies 36 genotypes by hybridization on a linear array of PGMY-generated amplicons with 34 type-specific probes for HPV-6, -11, -16, -18, -26, -31, -33, -35, -39, -40, -42, -45, -51, -53, -54, -55, -56, -58, -59, -61, -62, -64, -66, -67, -68, -69, -70, -71, -72, -73, -81, -83, -84, and -89, two probes for two subtypes of HPV-82, and one probe that cross-reacts with HPV-33, -35, -52, and -58 (8). A sample is thus considered positive for HPV-52 when it reacts with the HPV-52-cross-reactive probe but not with the HPV-33, -35, or -58 type-specific individual probes. However, LA-HPV cannot establish the presence of HPV-52 DNA in a sample reacting with the HPV-33/35/52/58-cross-reactive probe and with at least one of the HPV type-specific probe for HPV-33, -35, or -58. If these samples are considered positive for HPV-52, the prevalence of HPV-52 will be overestimated, and if they are considered negative, HPV-52 prevalence may be underestimated. HPV-52 is the seventh most frequently detected high-risk type in invasive cervical cancer worldwide (5) and causes 8.8% of low-grade and 2.5% of high-grade squamous intraepithelial lesions (4). It is thus imperative in epidemiological studies to be able to determine if HPV-52 is truly present in samples reacting with the HPV-52-cross-reactive probe and containing another type reacting with that probe. We present here the results of the validation of a novel real-time PCR assay for the rapid confirmation of HPV-52 infection in specimens positive with the HPV-52 cross-reacting probe in the LA-HPV test. Clinical specimens. Overall, 265 genital specimens collected from 95 women and 170 men participating in four studies described elsewhere (8, 11, 14, 16) were selected on the basis of previous testing of PGMY-generated amplicons with the cross-reacting HPV-33/35/52/58 probe and with conventional HPV-52 typing methods (see below). Anal swabs were collected in Preservcyt (Cytyc Corporation, Boxborough, MA) from 170 men in the HIPVIRG cohort with low-grade anal intraepithelial neoplasia (AIN) (n  74), high-grade AIN (n  56), AIN of unknown grade (n  4), or no AIN (n  36), and DNA was extracted as previously described (8). Cervical samples were collected and processed as previously described from * Corresponding author. Mailing address: De´partement de Microbiologie et Infectiologie, Hoˆpital Notre-Dame du Centre Hospitalier de l’Universite´ de Montre´al, 1560 Sherbrooke est, Montre´al (Que´bec), H2L 4M1, Canada. Phone: (514) 890-8000, ext. 25162. Fax: (514) 412-7512. E-mail: .
Published ahead of print on 26 September 2007. 3