Study design

In Sweden, all women aged 23-49 years are invited for cervical cancer screening at 3 year intervals and women aged 50-60 years at 5 year intervals. All women who are residents in Stockholm County, Sweden, who on their invitational smear had the cytological diagnoses ASCUS or LSIL between March 17, 2003, and Jan. 16, 2006, were included in a randomized health care policy as previously described, which was approved by the Ethical Review Board in Stockholm.

Sweden uses the old North American cytological terminology in which CIN grade I (CIN I) is also used as a cytological diagnosis. This corresponds closely to the LSIL in modern terminology and CIN I in cytology has therefore been substituted for LSIL throughout this paper. The policies compared were the referral of all women with ASCUS or LSIL for colposcopy and biopsy (previous policy) and HPV-based triaging referring all women with ASCUS or LSIL for a new visit with HPV testing using HCII.

All 15 obstetrics-gynecology clinics in Stockholm County were randomized to either colposcopy of all women (1567 women with ASCUS/LSIL) or to HPV triaging (1752 women with ASCUS/LSIL).

The present study focuses on the women randomized to the HPV triaging arm, in which 1600 samples could be analyzed with HCII. We extended the HPV testing to also include HPV typing of these samples. Because 5 samples were missing, we obtained HPV typing data on 1595 samples. The mean age of the women was 33 years (minimum 23 years, maximum 61 years). The HCII-positive women (n = 1154) were referred for a colposcopy-directed biopsy, and the HCII-negative women (n = 441) were referred for repeat cytology 12 months later. In the HPV triaging arm, a total of 1917 colposcopies were performed and 2766 biopsies were obtained. All histopathologies were interpreted on a routine practice basis.

HPV DNA testing

At enrollment, all samples were tested by HCII (Qiagen, Hilden, Germany), a pool-probe, signal amplification deoxyribonucleic acid (DNA) test that targets a group of 13 high-risk HPV genotypes (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). HCII does not provide information on the specific genotypes present. The samples were taken using the HCII DNA specimen collection kit (according to the manufacturer’s instructions) and stored frozen in specimen transport medium (STM). HCII was performed according to the manufacturer’s instructions.

DNA extraction

DNA from the samples stored in STM buffer were extracted using sodium dodecyl sulfate (SDS)/proteinase K. One hundred fifty microliters of lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, 4% SDS at pH 7.8) with the addition of 4% proteinase K was added to 50 μl of each sample and incubated at 37°C overnight. After the addition of 75 μL saturated NH ₄ Ac, the samples were centrifuged for 15 min at 16000 × g , the supernatant was transferred to a new tube, and the DNA was precipitated with 450 μL of absolute ethanol at –20°C for 30 minutes followed by 5 minutes of centrifugation at 16,000 × g . The DNA pellet was washed once with 250 μL 70 % ethanol, the dry pellet was dissolved in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA at pH 8.0) and stored at –20°C until analysis.

HPV DNA genotyping

PCR using the general primer pair GP 5+/bioGP 6+, was performed as previously described. Briefly, 1 μL of extracted DNA was added to the PCR master mix in a final volume of 25 μL. The PCR was performed in an Eppendorf Mastercycler epgradient (Eppendorf, Denmark), the first step at 94°C for 10 minutes was followed by 45 cycles of denaturing at 94°C for 1.5 minutes, annealing at 50°C for 2.0 seconds, and 40°C for 1.5 minutes, with a 7% ramp between 50°C and 40°C of 0.2°C/second, an extension step at 72°C for 2 minutes, and a terminal extension step at 72°C for 4 minutes. Positive controls were 10-fold dilutions, 1-0.01 ng/μL, of HPV 16 DNA purified from SiHa cells in TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, 10 ng/μl human placenta DNA, pH 8.0).

HPV detection and genotyping was performed using multiplex bead–based hybridization with Luminex technology as described by Schmitt et al. The probes for the HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 43, 45, 51, 52, 56, 58, 59, 66, 68, 70, 73, and 82 and an HPV 35 variant sequence (5′-CTG CTG TGT CTA CTA GTG A-3′) were used. The cutoff for positivity was set individually for each included HPV type to 2 times the mean mean fluorescent intensity of 12 negative water controls tested on each 96 well plate.

The samples were tested for amplifiability by real-time PCR amplification of the human β-globin gene as previously described using 0.2 μM of modified PCO ₃ and PCO ₄ primers (PCO ₃ 14-39F: 5′-ACACAACTGTGTTCACTAGCAACCTC-3′, PCO ₄ 123-103R: 5′-CCAACTTCATCCACGTTCACCT-3′) and 0.04 μM Taqman probe (β-globin 55-85: 5′-FAM-TGCACCTGACTCCTGAGGAGAAGTCTGC-TAMRA-3′) and 5 μL of sample in a 25 μL reaction.

Statistical analysis

Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using binomial logistic regression using the software R version 2.7.2 ( www.r-project.com ).