Tsiodras et al. BMC Cancer 2010, 10:53 http://www.biomedcentral.com/1471-2407/10/53 was used as positive control in the type-specific HPV PCR assays. The PCR procedure was repeated twice for every sample. A representative number of PCR products from each PCR assay was sequenced (ABI PRISM® 3730XL DNA Analyzers by Lark Technologies, Inc, UK) and the PCR products were confirmed. Statistical methods Sensitivity, specificity, positive and negative predictive values were calculated for cytological results and mole- cular HPV testing against the histological diagnosis of CIN2 and above or CIN3 and above. More specifically, we calculated values considering all cytology results ≥ ASCUS as positive, or all ≥ LSIL as positive or all ≥ HSIL as positive separately. For HC2 we calculated values for any positive result and for positive high risk results (all high risk infections independent of whether they were high risk only or mixed low-high risk infec- tions). For PCR we used either any positive result or positive results for HPV types 16, 18, 31 and 33 only. Positive and negative likelihood ratios were also calcu- lated [14]: likelihood ratios enable the comparison of diagnostic values of tests in a way independent of the prevalence of the disease. The PLR [sensitivity/(1 - spe- cificity)] corrects the true positive rate by the false posi- tive rate of a test while on the other hand NLR [(1 - sensitivity)/specificity] compares the probability of a negative test in persons with disease, compared to the probability of a negative test in persons without disease [14]. Finally, a Cohen’s kappa value was calculated for evaluating the agreement between the test results of HC2 and PCR methods. Results Cytological results During the 4 year period 1270 women were evaluated (mean age: 34.2 ± 12.1 years old). Table 1 depicts age associations with cytological findings and HPV detec- tion. The vast majority (n = 991, 78%) of the population examined were between 21 and 45 years old. Overall 241/1270 (19%) women were diagnosed with abnormal cytology (Table 1): more specifically, 101 (8%) women Page 3 of 8 exhibited ASC-US, 119 (9.4%) were diagnosed with LSIL, 10 (0.8%) with HSIL, 10 (0.8%) with squamous cell carcinoma and 1 (0.8%) with adenocarcinoma. HSIL or infiltrating carcinoma was the cytological diagnosis in 21/1270 (1.7%) samples. Reporting rates of ASC-US/ LSIL combined were 30.6% for women under 21 yrs old, 17.8% for the 22-30 yrs group, 17.7% for 30-45 yrs group, and then declined in older age groups (Table 1). HSIL+ rates increased with age, with 17/21 (81%) HSIL+ lesions diagnosed in women older than 30 years. Seven of the 11 (63.6%) invasive cancer cases were noted after 45 years of age (Table 1). Regarding HPV testing, PCR+ rates were approximately double those of ASC-US/LSIL combined for all age groups up until 65 years (Table 1). ThinPrep diagnosis in relation to HPV DNA detection by HC2 or PCR testing is depicted in Table 2. HPV was detected by HC2 in 260/1270 (20.4%) samples and by PCR in 397/1270 (31.3%) samples. The two tests gave highly concordant results (Cohen’s k value 0.691, 95% CI: 0.664 - 0.718). Discrepancies between HC2 and PCR were observed in 153 specimens. HC2 detected 8 speci- mens as being positive for HPV that were not detected by PCR (4 with ASCUS-all low risk HPV types; 4 with LSIL, 3 low and 1 high risk HPV types). PCR detected HPV in 145 specimens not detected by HC2, (80 with normal cytology, 45 with ASCUS, 16 with LSIL, 2 with HSIL, 1 with squamous cell carcinoma and 1 with ade- nocarcinoma). HC2 detected HPV in 17/21 (81%) while PCR detected HPV in 21/21 (100%) samples with HSIL+ cytology (Table 2). In the squamous cell carcinoma and adenocarcinoma categories 2/11 (18.2%) samples tested negative for HPV DNA with HC2, whereas none of these samples was negative with PCR (Table 2). In 21 cases HPV was not detected by either method: eleven cases with ASCUS and ten cases with LSIL. HC2 detected infection with high risk or mixed low- high risk HPV types in 40/101(39.6%) of ASCUS cases. PCR detected HPV 16, 18, 31, or 33 (single or multiple infections) in 31/86 (36%) of ASCUS cases. For LSIL the corresponding figures were 77/119 (64.7%) and 63/105 Table 1 Age associations with cytological findings and HPV isolation by PCR. Rates are given per total n of women per age group Age, yrs ≤ 21 22 - 30 30 - 45 45 - 65 > 65 Total HPV PCR data, n (%) Cytological data, n (%) HPV (-) 26(41.9) 353(67.6) 304(64.8) 141(75.4) 24(80) 873 HPV (+) 36 (58.1) 169 (32.4) 165 (35.2) 46 (24.6) 6 (20) 397 Total 62 522 469 187 30 1270 WNL 43(69.4) 425(81.4) 379(80.8) 156(83.4) 26(86.7) 1029 ASCUS 10 (16.1) 34(6.5) 43(9.2) 14(5) 0(0) 101 LSIL 9(14.5) 59(11.3) 40(8.5) 11(5.9) 0(0) 119 HgSIL 0(0) 3(0.6) 4(0.9) 3(1.6) 0(0) 10 SCC 0(0) 1(0.2) 3(0.6) 2(1.1) 4(13.3) 10 AdenoCa 0(0) 0(0) 0(0) 1(0.5) 0(0) 1 Total 62 522 469 187 30 1270