HPV testing and typing
The molecular biology methods have been described in detail elsewhere (3). Here we briefly report the adopted typing strategies.
The presence of high risk (HR) and low risk (LR) HPVs in cervical specimens was evaluated by Hybrid Capture II® (HCII) (Qiagen, Gaithersburg, USA) (12, 13, 14, 15, 16, 17) using probemix B, specific for 13 HR HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, and probemix A, specific for 5 LR HPV types: 6, 11, 42, 43 and 44 . The standard 1 pg/ml HPV DNA positivity threshold was adopted. HPV DNA testing was performed locally in 4 sites (one in Abruzzo, one in Campania and two in Rome) while samples collected in Cagliari and Catania were analysed in Florence (ISPO).
Typing procedures were centralized in Florence at the ISPO laboratory and all centres involved in the study sent HR or LR HPV positive samples there. The types included in IARC group 1 and 2a were classified as HR strains, the other types as LR ones .
DNA was extracted from 1.5 ml of samples in PreservCyt® solution and 200 μl of STM samples using QIAamp DNAMini Kit.
HCII HR HPV positive specimens were amplified and typed with "consensus High Risk HPV genotyping kit" (Qiagen), a system based on PCR with biotinylated GP5+/GP6+ primers , followed by reverse Line Blot for 18 HR HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73 and 82.
GP5+/6+PCR-negative and reverse Line Blot-negative samples were amplified for the β-globin gene using GH20-PC04 primers (268 bp amplicon lenght) to assess the integrity of DNA .
β-globin positive samples were re-typed with "INNO-LiPA HPV genotyping Extra Amp" (Innogenetics, Ghant, Belgium) following the manufacturer's instructions. This system targets 28 HR or LR HPV types: 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 69, 70, 71, 73, 74 and 82. β-globin negative samples were extracted and typed again.
The remaining un-typed samples (GP5+/6+PCR-positive, reverse Line Blot-negative and INNO-LiPA negative) were considered untyped HPV (HPV X).
HCII LR HPV positive specimens were typed using HPV6 and HPV11 specific primers . No LR type, other than 6 and 11, was searched for.
The prevalences of HR and LR HPV types by cytological class are reported. The proportion of HPV 16-18 and HPV 6-11-16-18 by cytological class are reported, including and excluding cases with co-infection of other HPV types. All analyses were performed separately for women under 35.
Co-infections of vaccine types with other types not included in vaccines are classified as follows: for HR vaccine types, HPV 16-18 alone (16, 18 or 16+18), HPV 16-18 + HR (HPV 16-18 + any non-vaccine high risk type independently from the presence of LR types), HPV 16-18 + LR (HPV 16-18 + any LR types, but without any co-infection of non-vaccine HR). Similarly, for LR vaccine types, HPV 6-11 alone (6, 11 or 6+11), HPV 6-11 + HR (HPV 6-11 + any non-vaccine high risk type, i.e. excluding 16-18, independently from the presence of other LR types), HPV 6-11 + LR (HPV 6-11 + any LR types, excluding any co-infection with HR types).
We estimated the age-adjusted Population Attributable Risk of Cytology abnormalities due to HPV vaccine type using a logistic model, according to the algorithm proposed by Fleiss , while 95% confidence intervals were computed according to the algorithm proposed by Rothman .
The analysis was performed taking into account the two stage sample using STATA 8 survey module .