engleski

Identification of HPV genotypes by PCR amplification and RFLP analysis among Croatian women

Cervical cancer was in the third place following breast and stomach cancer in Croatia in 1993. HPV infection as a leading cause of cervical cancer is an ideal prognostic factor for development of cervical neoplasia and cancer. The objectives of this study were to determine the HPV prevalence and HPV types among Croatian women, especially those with high-grade cervical intraepithelial neoplasia (CIN). The presence of HPV was evaluated in cervical samples collected at different gynaecological clinics from Zagreb (Croatia) from 1999 to 2001. HPV DNA was detected by polymerase chain reaction (PCR) using two sets of universal primers (MY09/MY11 and L1C1/L1C2) and type-specific primers for HPV 6/11, 16, 18, 31 and 33 as previously described (Grce M et al., Anticancer Res 21: 579 ; 2001). A subset of MY09/MY11-positive high-grade neoplasia (CIN III and CIS) samples was further analysed by restriction fragment length polymorphism (RFLP) using DdeI, DraI, PstI and Sau3AI restriction enzymes. Restriction fragments were analysed by agarose and polyacrilamide gel electophoresis. Out of 1669 specimens, 579 (34.7%) were negative, while 1090 (65.3%) were positive, of which 670 (61.5%) were determined by types-specific directed-PCR and 420 (38.5%) remained untyped (HPV X). The most frequently observed type was HPV 16 in 27.8% (303/1090) of HPV DNA positive samples. Out of 420 HPV X, 35 (8.3%) were further analysed by RFLP. Those 35 HPV X were chosen among MY09/MY11 positive samples with high-grade CIN in order to determine the prevalence of other supposed high risk HPV types other than 16, 18, 31 and 33. Fifteen out of 35 specimens were clearly defined according to RFLP pattern obtained with DdeI, DraI, PstI and Sau3AI enzymes, of which 5 corresponded to HPV 53, 2 to HPV 59, 2 to HPV 68 and one HPV 13, 33, 34, 39, 66 and MM9, respectively. Furthermore, according to RFLP patterns multiple infections with one defined and one undetermined HPV type were present in 5 different samples. High prevalence rate of HPV infection was found among Croatian women. High risk HPV, especially HPV 16 represent a significant health concern. However, infection with other risk types and HPV X should not be underestimated. HPV 53, although rare in a general population (Gallahan D et al., J Virol 63: 4911 ; 1994), seems to be associated with high-grade CIN among Croatian women. Our preliminary results should be confirmed on a larger number of samples.

papillomavirus; genotyping

nije evidentirano