In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5؉/6؉) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n ‫؍‬ 196) and biopsies of cervical premalignant lesions (n ‫؍‬ 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5؉/6؉ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5؉/6؉ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.High-risk human papillomavirus (HR-HPV) DNA has been shown to be present in 99.7% of cervical cancers worldwide (1,29), and the persistence of HR-HPV infection appears necessary for the development of cervical premalignant lesions and invasive cervical cancer (14,15,20,30). Therefore, HR-HPV testing may have implications for the clinical management of women with cervical lesions (25) and for primary screening for cervical cancer (17,20). In addition, several human papillomavirus (HPV)-targeted therapies have been or are being developed, and the first trials with prophylactic HPV vaccines are being conducted.To date, the detection of HPV genotypes has been done predominantly by L1 general-or consensus-primer PCR assays (3,6,12,16,24,26) and by the commercially available liquid hybridization assay Hybrid Capture 2 (HC2) (25). Generalprimer PCR assays enable the detection of a broad spectrum of mucosotropic HPV types, since the primers anneal to a highly homologous region of the HPV types spanning a polymorphic inner region, allowing specific HPV typing. Of these generalprimer PCR assays, the GP5ϩ/6ϩ and the MY09/11 PCR systems (3, 16) are the most frequently used and clinically evaluated ones. Despite the existence of more than 70 HPV genotypes, HPV testing for clinical purposes has been greatly simplified and facilitated by testing for all HR-HPV types simultaneously in one assay, i.e., HR-HPV group detection....

Before guidelines can be set for the use of high-risk human papillomavirus (HR HPV) testing in cervical cancer screening and vaccine preparation, age-related prevalence of HR HPV types in cytologically normal smears has to be known. Therefore, in a cross-sectional study the prevalence of 37 different HPV genotypes and putatively unidentified HPV types was determined in 3,305 cytologically normal cervical smears from the general female population (15-69 years of age) using an HPV general primer GP5+/bioGP6+ mediated PCR assay. Subsequently, HPV-positive cervical smears were typed for 19 HR and 18 low-risk (LR) HPVs with an enzyme immunoassay using HPV type-specific oligoprobes in cocktails and individually, respectively. Overall, -HR and -LR HPV prevalences appeared to be of 4.6%, 3.3%, and 1.0%, respectively. Twenty-six different HPV types were detected in the 152 HPV-positive samples, the most prevalent types being HPV 16, 31, and 18. With regard to age, a peak prevalence of 19.6% for all HPVs was found in women 25-29 years of age, which declined to a mean of 4.3% in women over 30 years. With regard cytologically normal cervical smears (n = 3, 011) of women participating in the population-based screening program in the Netherlands (30 to 60 years), all HR HPVs showed decreased occurrence with increasing age, whereas the prevalence of LR HPV types remained constant. We suggest that screening for abnormal cytology implies screening for HR HPV infections and the subsequent treatment results in a decline of HR HPV prevalence in contrast to LR HPV prevalence during the years of screening.

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Post-treatment monitoring by CADM1/MAL-methylation analysis identifies women with an increased risk of rCIN2/3. Our results confirm previous data indicating that CADM1/MAL-methylation analysis provides a high reassurance against cancer.