SESSIONE 2 Oral communications based on selected abstracts Conveners: S. Bruno, C.M. Mastroianni HCV NS3 Quasispecies in Liver and Plasma and Dynamics of Telaprevir-resistant Variants Assessed by UDPS in Breakthrough Patients: a Case Study B. Bartolini, M. Selleri, A.R. Garbuglia, E. Giombini, G. D’Offizi, M.R. Capobianchi National Institute for Infectious Diseases "L. Spallanzani", Rome, Italy Background Extended genetic quasispecies variability •High debate on the impact of preAntiviral drug existing variants carrying RAMs on resistance the outcome of antiviral treatment •Lack of knowledge of the role of reservoirs (liver...) in housing resistant variants that can be selected by drugs ??? In the liver: DAA resistance In the literature…… HEPATOLOGY, 2014 no compartmentalization between plasma and liver compartmentalization between plasma and liver Blood only may not adequately represent viruses replicating with in the liver AIM NS3 heterogeneity by UDPS in two HCV GT1a patients who underwent TVR+ pINF/RBV therapy and developed treatment failure baseline plasma and liver biopsy samples plasma samples during therapy plasma samples after therapy suspension The patients Stop Treatment 8 3 7 2,5 TVR 6 5 pINF/RBV 1,84 Pt2 2 1,49 4 HCV RNA logIU/ml 3 0,87 2 0,5 0,31 1 0 0 8 7 1 0,67 Diversity 3 HCV RNA 2,46 2,5 6 2 5 1,66 1,33 4 3 1,5 1 2 0,45 0,5 1 0 0 Baseline 1D 5D 15D 2M 3M 4M 1M after ST6M after ST Diversity (nt substitution/site)10-2 1,5 Pt22 No liver-plasma compartmentalization Liver Plasma pt2 pt22 Intra-patients diversity and prevalence of variants at position associated with resistance to TVR detected by UDPS in liver and plasma at baseline. Patient Viral Load (IU/ml) sample liver Pt2 Variants Observed (%) V36A (0.46) D168G (0.52) V36A (0.33) T54A (0.39) R155G (0.31) D168G (0.36) V36A (0.38) T54A (0.39) 0.00478 4.31x105 plasma liver Pt22 Diversity (nt substitution/site) 0.01836 0.0055 5.0 x106 plasma 0.0246 Dynamics of resistant quasispecies (Pt2) Variants Observed (%) baseline V36A (0.33) 1day of treatment V36A (0.34) 12 w after ST V36M,T T54A R155K,E (98.04), (0.39) (0.46) (98.56), (0.46) 24 w after ST V36M I132T R155K (8.47) (0.60) (98.94) 72 w after ST V36A T54A R155K (0.39) (0.44) (7.57) Conclusions No compartmentalization of HCV quasipecies between plasma and liver Drug resistance patterns at baseline did not significantly differ between plasma and liver De novo appearance of resistant population Tendency of RAM to disappear in FU Dynamics of disappearance to be studied with UDPS Longer FU and more patients to elucidate the issues TANKS TO Team of the Laboratory of Virology INMI “L.Spallanzani”, Rome Anna Rosa Garbuglia Marina Selleri and Maria R. Capobianchi you for the attention Prevalence of variants at positions associated with resistance to TVR detected by UDPS in plasma. Pt2 Viral Load (IU/ml) Pt22 Variants Observed (%) Viral Load (IU/ml) Variants Observed (%) baseline 4.31x105 V36A (0.33) 5.0 x106 V36A T54A (0.38) (0.39) 1day of treatment 3.95 x103 V36A (0.34) 3.40x103 V36A D168G (0.35) (0.36) x105 V36M I132T R155K (93.64) (1.01) (98.79) 2.49 x105 V36M,T T54A R155K D168A (86.27), (0.34) (0.35) (87.05) (0.47) 1 month from therapy stop 4.69 2.58x105 V36M,T T54A R155K,E (98.04), (0.39) (0.46) (98.56), (0.46) 6 month from therapy stop 6.07 x104 V36M I132T R155K (8.47) (0.60) (98.94) 18 month from therapy stop x105 V36A T54A R155K (0.39) (0.44) (7.57) 3 month from therapy stop 2.63 Intra-patients diversity and prevalence of variants at positions associated with resistance to TVR detected by Ultra Deep Sequencing in plasma. Patient Sample N° reads Viral Load (IU/ml) Diversity (nt substitution/site) baseline 3634 4.31x105 0.01836 V36A (0.33) - 1day of treatment 4655 3.95 x103 0.0031 V36A (0.34) - V36M,T (98.04), (0.39) T54A (0.46) R155K,E (98.56), (0.46) V36M (8.47) I132T (0.60) R155K (98.94) V36A (0.39) T54A (0.44) R155K (7.57) Variants Observed (%) Resistance Association (%) V36M+R155K 3 month from 3057 2.58x105 0.0149 (96.60) therapy stop Pt2 6 month from V36M+R155K 4156 6.07 x104 0.0067 therapy stop (8.42) 18 month from 4082 2.63 x105 0.0087 therapy stop - Intra-patients diversity and prevalence of variants at positions associated with resistance to TVR detected by Ultra Deep Sequencing in plasma. Patient Sample N° reads Viral Load (IU/ml) Diversity (nt substitution/site) Variants Observed (%) V36A baseline 7707 5.0 x106 4260 3.40x103 (0.38) - 0.0246 1day of T54A (0.39) V36A (0.35) - 0.0045 treatment D168G (0.36) V36M (93.64) 1 month from V36M+R155K 5059 Pt22 4.69 x105 0.0133 I132T (1.01) therapy stop (92.47) 6 month from 1158 therapy stop Resistance Association (%) 2.49 x105 R155K (98.79) V36M,T (86.27), (0.34) T54A (0.35) V36M+R155K R155K (87.05) (75.82) D168A (0.47) 0.0166 Background •High debate on the impact of preexisting variants carrying RAMs on the outcome of antiviral treatment •Lack of knowledge of the role of reservoirs (liver...) in housing resistant variants that can be selected by drugs Extended genetic variability quasispecies Antiviral drug resistance HEPATOLOGY, 2014 no compartmentalization between plasma and liver no compartmentalization between plasma and liver Blood only may not adequately represent viruses replicating with in the liver No liver-plasma compartmentalization Liver Plasma pt2 pt22
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