NGS in clinical Italian practice: impact of minor quasispecies on antiretroviral drug resistance and viral tropism AREVIR-GenaFor-Meeting Dr. Daniele Armenia Bonn 3th May 2012
Why use NGS in management of HIV-1 infection? Treatment begins Sensitive variant HIV RNA Level Minority resistant variant Time Incomplete Virological Failure Before starting suppression
Why use NGS in management of HIV-1 infection? Treatment begins Sensitive variant HIV RNA Level Minority resistant variant Time Incomplete Virological Failure Before starting suppression
Why use NGS in management of HIV-1 infection? Involved in resistance development of: • Low-mean genetic barrier Treatment begins ARVs such as NNRTI or INI • CCR5-Antagonists Sensitive variant HIV RNA Level (pre-existing minority X4 virus) Minority resistant variant Time Incomplete Virological Failure Before starting suppression
Why use NGS in management of HIV-1 infection? Involved in resistance development of: • Low-mean genetic barrier Treatment begins ARVs such as NNRTI or INI • CCR5-Antagonist Sensitive variant HIV RNA Level (pre-existing minority X4 NGS allows to detect hidden minority resistance variants and virus) Minority resistant theoretically to prevent their selection during treatment variant Time Incomplete Virological Failure Before starting suppression
Overview 1. To define new genetic markers of HIV that can predict the presence of transmitted RTIs drug resistant minority species. 2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.
Overview 1. To define new genetic markers of HIV that can predict the presence of transmitted RTIs drug resistant minority species. 2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.
Ultra-deep pyrosequencing (UDPS) of HIV-1 RT was performed using GS-FLX Roche, on plasma RNA from 40 drug-naive patients infected with HIV-1 subtype B without primary resistance detected by GRT.
In patients with L210M and T69S an higher number of minority drug resistance strains has been detected and these strains are present with a frequency higher than 1% Alteri et al, JAC 2011
In patients with L210M and T69S an higher number of minority drug resistance strains has been detected and these strains are present with a frequency higher than 1% This proof-of-concept study suggests the existence of genetic markers, detectable by routine testing, potentially acting as sentinel mutations of minority drug resistance
Overview 1. To define new genetic markers of HIV that can predict the presence of transmitted RTIs drug resistant minority species. 2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.
The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1–infected patients , who started a salvage raltegravir-containing regimen, were investigated. Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure
At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected Any statistically association neither with virologic response at 24-weeks nor with the development of resistant variants at failure was observed Armenia et al, JID 2011
UDPS Dynamics of Integrase Mutations During Raltegravir Treatment Armenia et al, JID 2011
Armenia et al, JID 2011
Resistance to raltegravir in integrase strand transfer inhibitor–naive patients remains today a rare event. Pathways of resistance at failure were not predicted by baseline mutations. Armenia et al, JID 2011
Overview 1. To define new genetic markers of HIV that can predict the presence of transmitted RTIs drug resistant minority species. 2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on viro-immunological response to MVC in clinical practice.
The Genotypic False Positive Rate Determined by Population V3-Sequencing Predicts the Burden of X4 Minority Quasispecies V Svicher, V Cento, G Rozera, I Abbate, MM Santoro, D Armenia, L Fabeni, G Palamara, A Latini, G Rizzardini, V Micheli, AR Buonomini, MP Trotta, A Antinori, M Andreoni, MR Capobianchi, F Ceccherini-Silberstein, CF Perno Manuscript submitted
V3 quasispecies composition at UDPS according to the false positive rate determined by population V3 sequencing 100.0 FPR of V3 sequences detected by UDPS 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 5.75% Cut-off 0.0 10-20 0-2 2-5 5-10 20-60 >60 (N=6) (N=5) (N=3) (N=4) (N=16) (N=12) FPR at population V3 sequencing Prevalence >20% Prevalence 10-20% R5 species X4 species Bulk V3 species Prevalence <10%
No X4 variants are detected by both UDPS and ESTA in all patients with FPR >60 100.0 FPR of V3 sequences detected by UDPS 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 5.75% Cut-off 0.0 10-20 0-2 2-5 5-10 20-60 >60 (N=6) (N=5) (N=3) (N=4) (N=16) (N=12) FPR at population V3 sequencing Prevalence >20% Prevalence 10-20% R5 species X4 species Bulk V3 species Prevalence <10%
The majority of patients with FPR ranging from 20 to 60 at population V3 sequencing harbour R5-species. 100.0 FPR of V3 sequences detected by UDPS 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 5.75% Cut-off 0.0 10-20 0-2 2-5 5-10 20-60 >60 (N=6) (N=5) (N=3) (N=4) (N=16) (N=12) FPR at population V3 sequencing Prevalence >20% Prevalence 10-20% R5 species X4 species Bulk V3 species Prevalence <10%
X4 species at a level >10% are present in all patients with FPR<5, and reach 90% of the entire population in patients with FPR<2 100.0 FPR of V3 sequences detected by UDPS 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 5.75% Cut-off 0.0 10-20 0-2 2-5 5-10 20-60 >60 (N=6) (N=5) (N=3) (N=4) (N=16) (N=12) FPR at population V3 sequencing Prevalence >20% Prevalence 10-20% R5 species X4 species Bulk V3 species Prevalence <10%
Overview 1. To define new genetic markers of HIV that can predict the presence of transmitted RTIs drug resistant minority species. 2. To investigate and quantify, using ultra-deep pyrosequencing (UDPS), the presence of integrase resistance mutations and to evaluate their impact on the virologic response to Raltegravir. 3. To define the potential correlation between FPR by population V3- genotyping and the burden of X4-species, detected by UDPS. 4. To evaluate the presence of hidden X4 species and their impact on virological response to MVC in clinical practice.
Analysis pipeline 15 Patients Plasma samples Baseline 454-junior and Sanger genotyping Starting Maraviroc R5-infected (Esta) + Sff output Fasta Geno2pheno Geno2pheno [coreceptor] [454] HIV-RNA monitoring to X4 tropism: evaluate virological X4 tropism: >2% of species with failure* FPR ≤ 10 % FPR ≤3.75 % *Virological failure: 2 consecutive HIV-1 RNA measurments >50copies/mL after at least 6 months of MVC pressure
Patient characteristics Overall patients 15 Age, Median (IQR) 41(37 -46) Male % 66.7 B (14) Subtype (N) BF (1) R5 Tropism (%) By phenotype (Esta) 100 By genotyping (geno2pheno FPR set at 10%) 86.6 Median (IQR) viral load at baseline 4.9 (4.5-4.3) (log 10 copies/ml) Median (IQR) CD4 cell count at baseline (cells/µl) 146 (120- 172) Median (IQR) GSS (rega 8.02) 2(2-3) Median (IQR) Number of experienced regimen 12(7 -15) Novel drugs co-admistered for the first time with MVC (%) DRV 13.3 ETR 6.7 RAL 33.3 T20 6.7
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