Primary resistance to Hepatitis B Virus in the treatment-naive Patients

Abstract

No: 1714 Presentation at ESCV 2015: Poster 2 Primary resistance to Hepatitis B Virus in the treatment-naive Patients M. Altindis1,∗, L. Demir1, I. Uslan2, Ozdemir Mehmet3, M. Baykan3, M. Kosecik4 1 Sakarya University School of Medicine, Dept of Clinical Microbiology, Sakarya, Turkey 2 Sakarya University School of Medicine, Dept of Gastroenterology, Sakarya, Turkey 3 Necmettin Erbakan Uni School of Medicine, Dept of clinical Microbiology, Konya, Turkey 4 Sakarya University School of Medicine, Dept of Pediatry, Sakarya, Turkey Background:Resistance to nucleos(t)ide analogues (NUC) is the main cause of NUC treatment failures leading to disease aggravation and complications. Our aim was to assess the prevalence of primary resistance to NUCs in a large cohort of treatment-naive patients infected with HBV by means of a highly sensitive method for the detection of minority viral populations. Methods: In this study, presence of resistance mutations in treatmentnaive individuals with chronic hepatitis B (CHB) was investigated in Sakarya University Hospital. A total of 59 patients (24 female, 35 male; age range: 19–66 years) diagnosed as CHB were enrolled in the study with informed consent from Gastroenterology, Sakarya anD Konya. All of the patients were negative for hepatitis C andD viruses and human immunodeficiency virus coinfections, and none had a history of interferon or NA treatment. HBV viral load(Montania® 4896, Bosphore® HBV Quantification Kit, Anatolia), HBV markers and hepatic enzymes in patients were determined via standardized commercial assays. For the detection of NA resistance mutations, a partial sequence of approximately 250 nucleotides, harboring the frequently-observed sites for NA resistance was amplified via nested PCR and characterized by direct sequencing of the amplicons(Bosphore® HBV Drug ResistanceMutation Sequencing Kit, Anatolia and Beckman Coulter CEQ 8000 Genetic Analysis System). The sequences were handled and interpreted for the presence ofmutations via various softwares and a web-based virtual phenotyping tool(CEQ 8000 Genetic Analysis System Version 9). Results: All circulating HBV strains were observed as genotype D(D1, D2, D3). At baseline, 59 patients harbored HBV resistanceassociated variants (RAV), generally in low proportions (0.3–20%). 26.9%, 9.7%, 1.6%, 1.6% of the patients displayed RAVs representing less than 1%, 1–5%, 5–10%, 10–20% of the viral quasispecies, respectively. 3 patients harbored RAVs that represented more than 20% of the quasispecies, including rtq215s, rtl91 and rtQ149K variant at 25%. None of the patients were found to harbor variants with both resistance and fitness mutations, which are generally selected on treatment at the sensitivity level of the technique. Conclusions: Primary HBV resistance to NUCs is frequent in treatment-naivepatients. These results further emphasize theneed for using first-line therapies including NUCs with a high barrier to resistance. http://dx.doi.org/10.1016/j.jcv.2015.07.273 Abstract No: 1725 Presentation at ESCV 2015: Poster 2 Development of an automated extraction system for Formalin-Fixed Paraffin Embedded (FFPE) tissue for the downstream detection of Human Papillomavirus (HPV) D. Guerendiain ∗, C. Moore, K. Cuschieri SHPVRL, NHS Lothian, Edinburgh, UK Background: Characterisation of FFPE biopsy material for HPV status is important for both epidemiological purposes and increasingly, for annotation of oropharyngeal squamous cell cancer (OPSCC). While manual extraction procedures for this application have been published–there is a lack of data on the development and performance of automated systems. The objective of this study was to develop and evaluate an automated extraction system for the detection of HPV nucleic acid using the NucliSENS® easyMAG®. Methods: A total of 60 samples were assessed: 30 FFPE sections related to high grade cervical intraepithelial neoplasia (CIN) lesions and 30 sections were derived from OPSCC. All samples had been extracted previously with manual silica columns (QIAamp DNA Mini Kit) and tested with the Optiplex HPV DNA assay (Diamex). Curled sections were obtained from each block with10 m thickness. Sampleswere extracted using the easyMAG® using a protocol adapted for FFPE material. DNA was tested using the Nanodrop® to evaluate the quantity and purity of DNA extracted. Agreement between the original HPV result associated with the manual extract and the results associatedwith the automated extract were assessed. Results: Nucleic acid yields ranged from 4.8 to 88ng/ l on extractsobtained fromtheeasyMAG® platform.At leastpartial concordance was observed in 93% in both cervical and OPSCC samples with an overall Kappa for HPV detection in the total sample (n=60) of 0.8049 (95% CI: 0.62–0.98). Specific kappas on HPV type-specific agreement will be presented. Conclusion:Our results indicate a high agreement between the easyMAG® and theQIAampDNAMiniKit® for the extractionofHPV DNA in FFPE samples derived from the oropharynx and the cervix This extraction method will reduce hands-on time – particularly for batch extraction. http://dx.doi.org/10.1016/j.jcv.2015.07.274