Materials. Since 1998, a total of 800 women, attending the Out-Patient Clinic of the Department of Gynecology and Obstetrics, Skopje, Republic of Macedonia, for routine gynecological check-ups, were tested for the presence of HPV. Informed consent was obtained from all patients. Clinical samples were taken by scraping the endo- and exo-cervix with a cytology brush. The specimens were smeared onto a slide, fixed with absolute ethanol, air-dried and sent to the laboratory for HPV testing. Fifteen women who showed the presence of a variant of HPV 66 were included in this study.

Methods. For routine HPV testing,DNA was isolated using the proteinase K digestion-phenol/chloroform extraction-ethanol precipitation method [11]; a negative control was included for every DNA isolation. HPV was detected by polymerase chain reaction (PCR) analysis using the MY09/MY11 consensus primers located in the L1 region of the virus [12]. The DNA quality of each specimen was ascertained by coamplification of b-globin gene fragment using the GH20/PC04 primers. AmpliTaq Gold polymerase (Applied BioSystems, Foster City, CA, USA) was used for all PC reactions. Positive and negative controls were included for every amplification. Aliquots of the PCR were run on a 1.5% agarose gel and analyzed under UV light following ethidium bromide staining. To confirm the specificity and to increase sensitivity, the PCR fragments were transferred to a nylon membrane and hybridized to a DIG-ddUTP labeled HPV group specific oligonucleotide probes (GP5+/GP6+). The detection was performed with Anti-DIG/AP conjugate and CDP* Star (Amersham Pharmacia Biotech UK Ltd., Little Chalfont, Buckinghamshire, UK) chemilluminiscence autoradiography. Samples with high viral load were typed by restriction fragment length polymorphism (RFLP) analysis of PCR amplified MY09/MY11 fragments using BamHI, DdeI, HaeIII, HinfI, PstI, RsaI and Sau3 AI restriction enzymes [13], while samples with low viral load were typed by dot-blot hybridization with nine type- specific oligonucleotide probes (HPV types 6, 11, 16, 18, 31, 33, 39, 58 and 66). The nucleotide sequence of the MY09/ MY11 PCR fragment of the HPV 66 variant was determined by fluorescent cycle sequencing on ABI PRISMO 310 (Applied BioSystems).