Improvement of HIV-1 resistance testing by proviral DNA analysis and Next Generation Sequencing. Nadine Lübke Institute of Virology Cologne - PDF

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Improvement of HIV-1 resistance testing by proviral DNA analysis and Next Generation Sequencing Nadine Lübke Institute of Virology Cologne 13th European HIV & Hepatitis Meeting 2015 Problems Majority of

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Improvement of HIV-1 resistance testing by proviral DNA analysis and Next Generation Sequencing Nadine Lübke Institute of Virology Cologne 13th European HIV & Hepatitis Meeting 2015 Problems Majority of samples from therapy-experienced patients with VL 500 copies/ml Low level (LLV) or undetectable viremia In most cases no results with plasma samples restricted detection limit of drug resistance mutations (DRMs) Sanger sequencing: 15-20% sensitivity cutoff 2 Possible Solutions proviral DNA (PBMCs) Unsuccessful testing with plasma RNA LLV or suppressed VL Problem VL often not known at timepoint of sample processing Cost and time intensive unnecessary repeats with plasma samples Total nucleic acid (tna= plasma RNA + proviral DNA) Effective resistance testing with unknown VL Saving cost and time 3 Comparison of mutation patterns 1. Viral RNA vs. proviral DNA of identical blood samples 2. Viral RNA vs. total NA of identical blood samples Sanger sequencing vs. NGS 4 Viral RNA vs. proviral DNA 69 samples of TE (n=46) and TN patients (n=23) of the RESINA cohort Paired viral RNA and proviral DNA were isolated PR and RT genes were amplified Sanger sequencing 47/69 samples (68%) presented DRMs in RNA and/or proviral DNA genotypes 5 DRMs detected in RNA only, DNA only or both 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% PR (n=47) RT (n=126) PRRT (n=173) only DNA only RNA RNA+DNA High concordance of the DRMs in viral RNA and proviral DNA (41.6%), especially the PI mutations (53.2%) Significant higher frequency of RTI mutations in RNA only (40.5%) compared to PI mutations (23.4%) (p=0.049) 6 Frequency of NRTI, NNRTI and PI mutations % samples with mutations (n=30) NRTI % samples with mutations (n=26) PI only RNA only DNA RNA+DNA NRTI resistance-associated mutations (n=75) PI resistance-associated mutations (n=47) % samples with mutations (n=29) NNRTI NNRTI resistance-associated mutations (n=51) 36 different resistance-associated positions in PR and RT 6/36 DRMs were more frequent in proviral DNA (NNRTI and PI mutations) 8/36 DRMs were more frequent in viral RNA (NRTI mutations) Overall high concordance of DRMs in RNA and DNA 7 Viral RNA vs. total NA 28 samples of TN patients of the RESINA cohort median VL=54,433 copies/ml (range ,000,000) Plasma viral RNA and total NA were isolated of each sample PR und RT genes were amplified Sequencing Sanger Sequencing (Sanger) Next Generation Sequencing (NGS) Illumina MiSeq (selected sensitivity cutoff 10%) 8 Prevalence of DRMs in viral RNA and tna viral RNA total NA P-value Sanger (n=27) DRMs Mean 1.19 ± ± 0.70 NGS (n=28) DRMs Mean 2.61 ± ± 1.68 P-value fold higher detection rate of DRMs in tna samples 2-fold higher detection rate of DRMs by NGS (p=0.0001) tna analyses provided a slightly increased DRM detection rate Significant higher DRM detection rate by NGS 9 DRMs detected in RNA only, tna only or both Sanger only RNA (n=2) NGS only RNA (n=31) 5% 27% only tna (n=12) RNA+tNA (n=30) 36% 26% only tna (n=44) RNA+tNA (n=42) 68% 38% Sanger: 6-fold higher detection rate of DRMs in tna vs. RNA only high concordance of DRMs in RNA and tna (68%) NGS: 1.5-fold higher detection rate of DRMs in tna vs. RNA only increased sensitivity of DRM detection independent of used nucleic acids 10 DRMs detected in RNA only or tna Sanger NGS only RNA (n=2) only RNA (n=31) 5% tna (n=42) 26% tna (n=86) 95% 74% Sanger: 95% of DRMs are detected by tna NGS: 74% of DRMs are detected by tna twice as many as detected by Sanger superior to Sanger 11 Summary and Conclusion RNA vs. DNA High concordance of DRMs in viral RNA and proviral DNA Proviral DNA resistance testing could help in cases of unsuccessful RNA genotyping (LLV, suppressed VL) RNA vs. tna Total NA provided an increased DRM detection rate Total NA could be an alternative to plasma RNA analyses Sanger vs. NGS NGS significantly increased the resistance information NGS could be an alternative in routine diagnostic 12 Thanks to Institute of Virology, University of Cologne Rolf Kaiser Sarah Reinartz Elena Knops Veronica Di Cristanziano Maria Neumann-Fraune Eugen Schülter Claudia Müller Dörte Hammerschmidt Ramona Gilles Saleta Sierra-Aragon Eva Heger RESINA Co-operation partner Thomas Lengauer, MPI of Informatics, Saarbrücken Martin Däumer, Alex Thielen, Institut für Immungenetik, Kaiserslautern Jens Verheyen, Virologie Essen RESINA Funding BMG: IIA AUK375 Björn Jensen, Dept. Gastroeneterology, Hepatology & Infectiology, University of Duesseldorf Gerd Fäktenheuer, Dept. Internal Medicine I, University of Cologne Stefan Esser, Dept. of Dermatology, University of Essen Jürgen Rockstroh, Dept. Internal Medicine I, University of Bonn Mark Oette, Dept. Gastroenterologie, Krankenhaus der Augustinerinnen, Cologne Stefan Scholten, Privat Practice, Köln Patrick Braun, Heribert Knechten, PZB Aachen 13
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