<i>In-Depth</i> Analysis of HA and NS1 Genes in A(H1N1)pdm09 Infected Patients - Fig 2

<p>Frequency of minority variants of HA (panel A) and NS1 (panel B) proteins in influenza A(H1N1)pdm09 virus present in the nasopharyngeal swabs of 12 out of 13 patients of the study. Each patient is identified by a color code. The amino acid positions are referred to A/California/07/2009(H1N1), adopted as reference for this analysis (H1 numbering excluding the signal peptide).</p

Correlation between nucleotide diversity of NS1 and viral load.

<p>On x-axis, NS1 nucleotide diversity, expressed as Nucleotide Substitution/Site X 10⁻⁴; on y-axis, viral load expressed as log₁₀ copies/mL.</p

Demographic, Clinical and Virological Features of the Study Patients.

<p>Demographic, Clinical and Virological Features of the Study Patients.</p

IFNAR-1 mRNA levels before and after treatment with IFN-alpha in PBMC from naive HCV-infected patients.

<p>Total cellular RNA was extracted and reverse-transcribed from PBMC of naive HCV-infected patients carrying different IL-28B rs12979869 genotypes CC, TT and CT before (<b>Panel A</b>) and after 3 h of exposure to 10³ IU/ml IFN-alpha (<b>Panel B</b>), then mRNA levels for IFNAR-1 were measured. Results are expressed as ratio to beta-actin (median, IQR).</p

Levels of IFNAR-1 mRNA in PBMC from the HCV-infected naïve subjects after 3h of exposure to 10³ IU/ml IFN-alpha2b <i>in vitro</i>, according to their <i>IFNL3</i> and <i>IFNL4</i> genotype combinations.

<p>Group 1: <i>IFNL3</i> CC and <i>IFNL4</i> TT/TT (<i>IFNL3</i> favourable, <i>IFNL4</i> favourable) n = 6; Group 2: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/TT (<i>IFNL3</i> unfavourable and <i>IFNL4</i> favourable) n = 11; Group 3: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/ΔG or ΔG/ΔG (<i>IFNL3</i> and <i>IFNL4</i> unfavourable) n = 11. The results are expressed as ratio to beta-actin, after subtraction of values from unexposed cultures (median, IQR).</p

Characteristics of 32 treatment naive HCV-infected patients included in the study.

<p>Characteristics of 32 treatment naive HCV-infected patients included in the study.</p

Levels of IFNAR-1 mRNA in PBMC from the HCV-infected naïve subjects grouped according to their <i>IFNL3</i> and <i>IFNL4</i> genotype combinations.

<p>Panel A. Group 1: <i>IFNL3</i> CC and <i>IFNL4</i> TT/TT (<i>IFNL3</i> favourable, <i>IFNL4</i> favourable), n = 8; Group 2: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/TT (<i>IFNL3</i> unfavourable and <i>IFNL4</i> favourable) n = 10; Group 3: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/ΔG or ΔG/ΔG (<i>IFNL3</i> and <i>IFNL4</i> unfavourable) n = 14. The results are expressed as ratio to beta-actin (median, IQR). Levels of IP10 mRNA in PBMC from the various groups after 3h of exposure to 10³ IU/ml IFN-alpha2b <i>in vitro</i>, according to their <i>IFNL3</i> and <i>IFNL4</i> genotype combinations. Panel B. Group 1: <i>IFNL3</i> CC and <i>IFNL4</i> TT/TT (<i>IFNL3</i> favourable, <i>IFNL4</i> favourable) n = 6; Group 2: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/TT (IFNL3 unfavourable and <i>IFNL4</i> favourable) n = 11; Group 3: <i>IFNL3</i> CT or TT and <i>IFNL4</i> TT/ΔG or ΔG/ΔG (<i>IFNL3</i> and <i>IFNL4</i> unfavourable) n = 11. The results are expressed as ratio to beta-actin, after subtraction of values from unexposed cultures (median, IQR). The range of IP10 mRNA levels in unexposed PBMC cultures was 0,375 to 0,967.</p

Levels of IFNAR-1 mRNA in PBMC from HCV-infected naїve subjects carrying <i>IFNL4</i> ss469415590 TT/TT genotype vs patients carrying the ΔG allele.

<p>Results are expressed as ratio to beta-actin. The horizontal bar indicates median.</p

Time dependent induction of IFNAR-1 mRNA levels following treatment with IFN-lambda in PBMC from healthy donors.

<p>PBMC were exposed to control medium (▪) 10 ng/mL (▪) and 100 ng/mL (□) of IFN-lambda, then mRNA levels for IFNAR-1 were measured at different time points (0, 3, 6, 12 and 24 h). Results are expressed as ratio to beta-actin. Results from one representative experiment performed on PBMC from two healthy donors with IL-28B rs12979860 CC (<b>Panel A</b>) and TT (<b>Panel B</b>) genotype are shown.</p

Ultrasensitive HCV RNA Quantification in Antiviral Triple Therapy: New Insight on Viral Clearance Dynamics and Treatment Outcome Predictors

<div><p>Objectives</p><p>Identifying the predictive factors of Sustained Virological Response (SVR) represents an important challenge in new interferon-based DAA therapies. Here, we analyzed the kinetics of antiviral response associated with a triple drug regimen, and the association between negative residual viral load at different time points during treatment.</p><p>Methods</p><p>Twenty-three HCV genotype 1 (GT 1a n = 11; GT1b n = 12) infected patients were included in the study. Linear Discriminant Analysis (LDA) was used to establish possible association between HCV RNA values at days 1 and 4 from start of therapy and SVR. Principal component analysis (PCA) was applied to analyze the correlation between HCV RNA slope and SVR. A ultrasensitive (US) method was established to measure the residual HCV viral load in those samples which resulted “detected <12IU/ml” or undetectable with ABBOTT standard assay, and was retrospectively used on samples collected at different time points to establish its predictive power for SVR.</p><p>Results</p><p>According to LDA, there was no association between SVR and viral kinetics neither at time points earlier than 1 week (days 1 and 4) after therapy initiation nor later. The slopes were not relevant for classifying patients as SVR or no-SVR. No significant differences were observed in the median HCV RNA values at T0 among SVR and no-SVR patients. HCV RNA values with US protocol (LOD 1.2 IU/ml) after 1 month of therapy were considered; the area under the ROC curve was 0.70. Overall, PPV and NPV of undetectable HCV RNA with the US method for SVR was 100% and 46.7%, respectively; sensitivity and specificity were 38.4% and 100% respectively.</p><p>Conclusion</p><p>HCV RNA “not detected” by the US method after 1 month of treatment is predictive of SVR in first generation Protease inhibitor (PI)-based triple therapy. The US method could have clinical utility for advanced monitoring of virological response in new interferon based DAA combination regimens.</p></div