Performance Evaluation of Manual and Automated (MagNA Pure) Nucleic Acid Isolation in HPV Detection and Genotyping Using Roche Linear Array HPV Test

Nucleic acids of human papillomavirus (HPV) isolated by manual extraction method (AmpliLute) and automated MagNA pure system were compared and evaluated with cytohistological findings in 253 women. The concordance level between AmpliLute and MagNA was very good 93.3% (κ = 0.864, P < .0001). Overall HPVpositivity detected by AmpliLute was 57.3% (30.4% as single and 27% as multiple infections) in contrast to MagNA 54.5% (32% and 23%, resp.). Discrepant results observed in 25 cases: 11 MagNA(−)/AmpliLute(+), 10 of which had positive histology; 5 MagNA(+)/AmpliLute(−) with negative histology; 8 MagNA(+)/AmpliLute(+): in 7 of which AmpliLute detected extra HPV genotypes and 1 MagNA(invalid)/AmpliLute(+) with positive histology. Both methods performed well when compared against cytological (area under curve (AUC) of AmpliLute 0.712 versus 0.672 of MagNA) and histological diagnoses (AUC of AmpliLute 0.935 versus 0.877 of MagNA), with AmpliLute showing a slightly predominance over MagNA. However, higher sensitivities, specificities, and positive/negative predictive values were obtained by AmpliLute

Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women

The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 (n = 104), HPV 31 (n = 40), HPV 33 (n = 7), HPV 18 (n = 5), and HPV 45 (n = 4) were typed by microarrays method, amplified by PCR then sequenced and phylogenetically analyzed. For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk

Promoter Methylation of p16INK4A, hMLH1, and MGMT in Liquid-Based Cervical Cytology Samples Compared with Clinicopathological Findings and HPV Presence

Cervical cancer is a common cancer inflicting women worldwide. Even though, persistent infection with oncogenic Human Papillomavirus (HPV) types is considered the most important risk factor for cervical cancer development, less than 5% of women with HPV will eventually develop cervical cancer supporting that other molecular events, like methylation-dependent inactivation of tumor suppressor genes, may cocontribute in cervical carcinogenesis. We analyzed promoter methylation of three candidate genes (p16, MGMT, and hMLH1) in 403 liquid-based cytology samples. Methylation was commonly identified in both benign and pathologic samples and correlated with higher lesion grade determined by cytological, colposcopical, or histological findings, with HPV DNA and mRNA positivity of specific HPV types and p16INK4A protein expression. Overall accuracy of methylation is much lower than traditional diagnostic tests ranking it as an ancillary technique with more data needed to identify the exact value of methylation status in cervical carcinogenesis

Hybrid capture vs. PCR screening of cervical human papilloma virus infections. Cytological and histological associations in 1270 women

<p>Abstract</p> <p>Background</p> <p>We evaluated two molecular methods of HPV detection and their correlation with cytological and histological diagnosis in a large sample of Greek women.</p> <p>Methods</p> <p>All women with liquid-based cytology performed at a University Hospital between 2000 and 2003 were included. The Hybrid Capture 2 (HC2) kit and in house Polymerase Chain Reaction (PCR) were used for HPV DNA detection. Cervical biopsy was performed for women with ASCUS+ cytology, HPV detection, or abnormal colposcopy. Positive (PLR) and negative (NLR) likelihood ratios were calculated for cytology and HPV molecular testing for the prediction of CIN2 and greater histology.</p> <p>Results</p> <p>Of the 1270 women evaluated 241 (18.5%) had abnormal cytology. Cytology diagnosed high-grade squamous intraepithelial lesion (HSIL) or invasive carcinoma in 21(1.7%) cases whereas 26 (2%) women had CIN2+ or greater histology. PCR detected HPV in 397/1270 (31.3%) and HC2 in 260/1270 (20.4%) samples. Both molecular tests exhibited high reproducibility (Cohen's kappa value 0.691, 95% CI: 0.664 - 0.718). Positive likelihood ratios (PLR) of 9.4, 3.8 and 3.4 and negative likelihood ratios of 0.13, 0.21, and 0 were noted for ≥ LSIL, any positive HC2 or any positive PCR-HPV testing, for predicting CIN2+ histology, respectively. All CIN 3+ lesions harbored high risk oncogenic HPV type infections.</p> <p>Conclusions</p> <p>HPV infection was found in a large proportion of this population and was associated with CIN 2/3 lesions and infiltrating carcinomas. Thin prep testing and HPV detection by HC2 or PCR performed very well with regards to identifying high grade lesions in an environment with experienced examiners.</p

Genetic Variability and Phylogeny of High Risk HPV Type 16, 18, 31, 33 and 45 L1 Gene in Greek Women

The present study explores nucleotide variability, phylogeny and association with cervical neoplasia in high risk HPV types 16, 18, 31, 33 and 45 collected from Greek women. Of the 1894 women undergoing routine cervical cytology examination, 160 samples test positive for single infections of HPV type 16 (n = 104), HPV 31 (n = 40), HPV 33 (n = 7), HPV 18 (n = 5), and HPV 45 (n = 4) were typed by microarrays method, amplified by PCR then sequenced and phylogenetically analyzed. For HPV 16, 9 variants with nucleotide variations were included into the study. For HPV 31, 33, 18 and 45, nucleotide variations were identified in 6, 4, 2 and 3 variants, respectively. The Bayesian inference and Maximum Parsimony methods were used in order to construct the phylogenetic trees. When types were analyzed independently HPV 16 (European and non-European) and HPV 18 (African and non-African) formed distinct clades. The genomic characterization of HPV variants will be important for illuminating the geographical relatedness and biological differences and for the determination of their risk

mRNA and DNA detection of human papillomaviruses in women of all ages attending two colposcopy clinics.

OBJECTIVE: HPV infection is a common finding, especially in young women while the majority of infections are cleared within a short time interval. The aim of this study was to examine the efficacy of HPV DNA and mRNA testing in a population attending colposcopy units of two University hospitals. METHODS: 1173 liquid based cervical samples from two colposcopy clinics were tested for HPV DNA positivity using a commercial typing kit and HPV E6/E7 mRNA positivity with a flow cytometry based commercial kit. Statistic measures were calculated for both molecular tests and morphological cytology and colposcopy diagnosis according to histology results. RESULTS: HPV DNA, high-risk HPV DNA, HPV16 or 18 DNA and HPV mRNA was detected in 55.5%, 50.6%, 20.1% and 29.7% of the cervical smears respectively. Concordance between the DNA and the mRNA test was 71.6% with their differences being statistically significant. Both tests' positivity increased significantly as lesion grade progressed and both displayed higher positivity rates in samples from women under 30 years old. mRNA testing displayed similar NPV, slightly lower sensitivity but significantly higher specificity and PPV than DNA testing, except only when DNA positivity for either HPV16 or 18 was used. CONCLUSIONS: Overall mRNA testing displayed higher clinical efficacy than DNA testing, either when used as a reflex test or as an ancillary test combined with morphology. Due to enhanced specificity of mRNA testing and its comparable sensitivity in ages under 25 or 30 years old, induction of mRNA testing in young women could be feasible if a randomized trial verifies these results

Promoter Methylation of p16 INK4A , hMLH1, and MGMT in Liquid-Based Cervical Cytology Samples Compared with Clinicopathological Findings and HPV Presence

Cervical cancer is a common cancer inflicting women worldwide. Even though, persistent infection with oncogenic Human Papillomavirus (HPV) types is considered the most important risk factor for cervical cancer development, less than 5% of women with HPV will eventually develop cervical cancer supporting that other molecular events, like methylation-dependent inactivation of tumor suppressor genes, may cocontribute in cervical carcinogenesis. We analyzed promoter methylation of three candidate genes (p16, MGMT, and hMLH1) in 403 liquid-based cytology samples. Methylation was commonly identified in both benign and pathologic samples and correlated with higher lesion grade determined by cytological, colposcopical, or histological findings, with HPV DNA and mRNA positivity of specific HPV types and p16 INK4A protein expression. Overall accuracy of methylation is much lower than traditional diagnostic tests ranking it as an ancillary technique with more data needed to identify the exact value of methylation status in cervical carcinogenesis