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The residual exfoliated cells were stored at ?20��C until DNA extraction. The final diagnosis of the women with abnormal cytology was made by histological evaluation on colposcopically directed biopsies. Outcome was defined as normal (normal cytology and/or negative biopsy), CIN1 or CIN2 or worse (CIN2+) on cytological/histological examination [23]. Both diagnoses (cytological and histological) were confirmed by experienced pathologists. All women provided voluntary written informed consent for the tests and answered a questionnaire. The study protocol was approved Histone demethylase by the sponsored competent ethical review committees. DNA was extracted manually from all cervical specimens. Cell suspensions were digested with 100?��g/mL proteinase?K for 3?h at 56��C, and purified by spin-column chromatography. Nucleic acids were resuspended in a final volume of 100?��L and stored at ?20��C until use for PCR Enzalutamide amplification. Genotyping was performed by using the Clinical Array HPV Assay (now CLART HPV 2; Genomica, Madrid, Spain), according to the manufacturer��s procedures. This methodology uses biotinylated primers that amplify a 450-bp fragment within the HPV L1 region. Co-amplification of an 892-bp region of the cystic fibrosis transmembrane conductance regulator gene and a 1202-bp fragment of a transformed plasmid provides a control to ensure DNA extraction adequacy and PCR efficiency. Amplicons are detected by hybridization in a low-density microarray containing triplicate DNA probes specific to 35 types (HPV?6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 61, 62, 66, 68, 70, 71, 72, 73, 81, 82, 83, 84, 85, and 89). Semiquantitative results can be obtained in an automatic reader. In this study, HPV?16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82 were considered as HR HPV (including probable and possible HR types), and HPV?6, 11, 40, 42, 43, 44, 54, 61, 62, 71, 72, 81, 83, 84, 85 and 89 as LR HPV (including unclassified types) [2,24]. To determine the prevalence of HR HPV and LR HPV types, cases were counted more than once if they harboured a multiple infection with a mixture of both. The prevalence of individual HPV types was determined as they appeared in single or in multiple infections. Multiple HPV infection was Venetoclax mw defined as two or more HPV types detected. For multiple HPV infections, the proportion was assessed in relation to severity of lesion, and compared with data from women harbouring a single HPV infection. Data were analysed using the chi-squared test, and, when appropriate, Fisher��s exact test to calculate all p-values. The ORs, together with 95%?CIs, were computed with the use of 2?��?2 contingency tables. All analyses were performed with SPSS version?17.0 software. Differences were considered to be statistically significant when p-values were