Sequence analysis of human papillomavirus (HPV) general primer GP5/6 mediated PCR products revealed the presence of short highly conserved sequences adjacent to the 3' ends of both primers. Part of these sequences was used to elongate GP5 and GP6 at their 3' ends to generate the primers GP5 + and GP6+, respectively. Compared with the GP5/6 PCR, GP5 +/ 6+ specific PCR on 22 cloned mucosotropic HPVs revealed an improved HPV detection, reflected by a 10-to 100-fold higher sensitivity and a markedly increased signal to background ratio, especially at the gel level. As determined on purified DNA, the sensitivity of this GP5 +/6 + based assay was at the femtogram level for those HPV genotypes which match strongly with the primers (e.g. HPV-16) and at the picogram level for HPV types (e.g. HPV-39 and -51) having four or more mismatches with one or both primers. Application of both methods on 264 cervical scrapes of a cohort of women participating in a prospective follow-up study revealed an increase of total HPV positivity from 39 % (GP5/6 PCR) to 43 % (GP5 +/6 + PCR) of the scrapes. Additional HPV typing by PCR specific for the HPV-6, -11, -16, -18, -31 and -33 revealed that all GP5+/6+ PCR positive cases which were negative by GP5/6 PCR (n = 12) contained HPV types different from these six types. These data indicate that the GP5 +/6+ PCR method provides an increased detection level mainly of uncommon, apparently poorly matched HPV types in cervical scrapes and most likely in the enlargement of the spectrum of HPVs detectable by this assay.

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....

Objectives To investigate the role of human papillomavirus (HPV) in the development of cervical neoplasia in women with no previous cervical cytological abnormalities; whether the presence of virus DNA predicts development of squamous intraepithelial lesion; and whether the risk of incident squamous intraepithelial lesions differs with repeated detection of the same HPV type versus repeated detection of different types. Design Population based prospective cohort study. Setting General population in Copenhagen, Denmark. Participants 10 758 women aged 20-29 years followed up for development of cervical cytological abnormalities; 370 incident cases were detected (40 with atypical squamous cells of undetermined significance, 165 with low grade squamous intraepithelial lesions, 165 with high grade squamous intraepithelial lesions). Main outcome measures Results of cervical smear tests and cervical swabs at enrolment and at the second examination about two years later. Results Compared with women who were negative for human papillomavirus at enrolment, those with positive results had a significantly increased risk at follow up of having atypical cells (odds ratio 3.2, 95% confidence interval 1.3 to 7.9), low grade lesions (7.5, 4.8 to 11.7), or high grade lesions (25.8, 15.3 to 43.6). Similarly, women who were positive for HPV at the second examination had a strongly increased risk of low (34.3, 17.6 to 67.0) and high grade lesions (60.7, 25.5 to 144.0). For high grade lesions the risk was strongly increased if the same virus type was present at both examinations (813.0, 168.2 to 3229.2). Conclusions Infection with human papillomavirus precedes the development of low and high grade squamous intraepithelial lesions. For high grade lesions the risk is greatest in women positive for the same type of HPV on repeated testing.

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5؉/6؉ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed >CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age ‫؍‬ 42.8 years; range ‫؍‬ 29 -61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3.6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), <0.1% suspected for invasive cancer (hrHPV positive 100.0%). In the Control Group 96.5% of the women had normal cytology, 2.4% BMD and 0.8% >BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, <0.1% suspected for invasive cancer. The presence of hrHPV was age-dependent, decreasing from 12.0% at 29 -33 years to 2.4% at 59 -61 years. Among women with a positive hrHPV test, the prevalence of BMD was age-dependent ranging from 20.2% at 29 -33 years to 7.8% at 54 -58 years. In contrast, the risk of >BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5؉/6؉ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.

Human papillomavirus is the principal risk factor associated with cervical cancer, the most common malignancy among women in Colombia. We conducted a survey, aiming to report type specific prevalence and determinants of human papillomavirus infection in women with normal cytology. A total of 1859 women from Bogota, Colombia were interviewed and tested for human papillomavirus using a general primer GP5+/GP6+ mediated PCR -EIA. The overall HPV DNA prevalence was 14.8%; 9% of the women were infected by high risk types, 3.1% by low risk types, 2.3% by both high risk/low risk types and 0.4% by uncharacterized types (human papillomavirus X). Thirty-two different human papillomavirus types were detected, being human papillomavirus 16, 58, 56, 81(CP8304) and 18 the most common types. The human papillomavirus prevalence was 26.1% among women younger than 20 years, 2.3% in women aged 45 -54 years, and 13.2% in women aged 55 years or more. For low risk types the highest peak of prevalence was observed in women aged 55 years or more. Compared to women aged 35 -44 years, women aged less than 20 years had a 10-fold increased risk of having multiple infections. Besides age, there was a positive association between the risk of human papillomavirus infection and number of regular sexual partners and oral contraceptive use. In women aged below 25 years, high educational level and having had casual sexual partners predicted infection risk. In conclusion, there was a broad diversity of human papillomavirus infections with high risk types being the most common types detected. In this population multiplicity of sexual partners and, among young women, high educational level and casual sexual partners seem to determine risk.