Hepatitis C Virus (HCV) Genotype 1 Subtype Identification in New HCV Drug Development and Future Clinical Practice

International audienceBACKGROUND: With the development of new specific inhibitors of hepatitis C virus (HCV) enzymes and functions that may yield different antiviral responses and resistance profiles according to the HCV subtype, correct HCV genotype 1 subtype identification is mandatory in clinical trials for stratification and interpretation purposes and will likely become necessary in future clinical practice. The goal of this study was to identify the appropriate molecular tool(s) for accurate HCV genotype 1 subtype determination. METHODOLOGY/PRINCIPAL FINDINGS: A large cohort of 500 treatment-naïve patients eligible for HCV drug trials and infected with either subtype 1a or 1b was studied. Methods based on the sole analysis of the 5' non-coding region (5'NCR) by sequence analysis or reverse hybridization failed to correctly identify HCV subtype 1a in 22.8%-29.5% of cases, and HCV subtype 1b in 9.5%-8.7% of cases. Natural polymorphisms at positions 107, 204 and/or 243 were responsible for mis-subtyping with these methods. A real-time PCR method using genotype- and subtype-specific primers and probes located in both the 5'NCR and the NS5B-coding region failed to correctly identify HCV genotype 1 subtype in approximately 10% of cases. The second-generation line probe assay, a reverse hybridization assay that uses probes targeting both the 5'NCR and core-coding region, correctly identified HCV subtypes 1a and 1b in more than 99% of cases. CONCLUSIONS/SIGNIFICANCE: In the context of new HCV drug development, HCV genotyping methods based on the exclusive analysis of the 5'NCR should be avoided. The second-generation line probe assay is currently the best commercial assay for determination of HCV genotype 1 subtypes 1a and 1b in clinical trials and practice

HIV-1/M+O INTERGROUP DUAL INFECTIONS AND ASSOCIATED HIV-MO RECOMBINANTS IN FRANCE FROM 2004 TO 2014

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Multicenter clinical comparative evaluation of Alinity m HIV-1 assay performance.

Abstract Background Accurate, rapid detection of HIV-1 RNA is critical for early diagnosis, treatment decision making, and long-term management of HIV-1 infection. Objective We evaluated the diagnostic performance of the Alinity m HIV-1 assay, which uses a dual target/dual probe design against highly conserved target regions of the HIV-1 genome and is run on the fully automated Alinity m platform. Study design This was an international, multisite study that compared the diagnostic performance of the Alinity m HIV-1 assay to four commercially available HIV-1 assays routinely used in nine independent clinical laboratories. Alinity m HIV-1 assay precision, detectability, and reproducibility was compared across four study sites. Results The Alinity m HIV-1 assay produced comparable results to currently available HIV-1 assays (correlation coefficient >0.995), with an overall bias of -0.1 to 0.10 Log10 copies/mL. The Alinity m HIV-1 assay and its predecessor m2000 HIV-1 assay demonstrated comparable detection of 16 different HIV-1 subtypes (R2 = 0.956). A high level of agreement (>88 %) between all HIV-1 assays was seen near clinical decision points of 1.7 Log10 copies/mL (50 copies/mL) and 2.0 Log10 copies/mL (200 copies/mL). Alinity m HIV-1 assay precision was 0.08 and 0.21 Log10 copies/mL at VLs of 1000 and 50 copies/mL, respectively, with a high level of detectability (≥97 % hit rate) and reproducibility across sites. Conclusions The Alinity m HIV-1 assay provides comparable diagnostic accuracy to current HIV-1 assays, and when run on the Alinity m system, has the capacity to shorten the time between diagnosis and treatment

Performance of the Abbott Real-Time PCR Assay Using m2000sp and m2000rt for Hepatitis C Virus RNA Quantification▿

Quantification of hepatitis C virus (HCV) RNA is essential for the everyday management of chronic hepatitis C therapy. “Real-time” PCR techniques are potentially more sensitive than classical PCR techniques, are not prone to carryover contamination, and have a consistently wider dynamic range of quantification. Thus, they are rapidly replacing other technologies for routine quantification of HCV RNA. We extensively evaluated the intrinsic characteristics and clinical performance of the m2000sp-m2000rt Abbott real-time PCR platform for HCV RNA quantification. The study shows that the m2000sp-m2000rt platform is sensitive, specific, and precise; that the results are reproducible; and that the platform has a broad dynamic range of quantification. When comparing HCV RNA levels measured in the same individuals with the m2000sp-m2000rt platform and the third-generation branched-DNA assay, a trend toward a modest overestimation of HCV RNA levels was observed in the m2000sp-m2000rt platform in all genotypes except genotype 5. The differences, however, were unlikely to have any impact in clinical practice. In conclusion, our study shows that the Abbott m2000 real-time PCR system for HCV RNA quantification is sensitive, specific, and precise; that the results are reproducible; and that the platform's broad dynamic range of quantification is well suited to HCV RNA monitoring in the clinical setting

Performance of the Cobas AmpliPrep/Cobas TaqMan Real-Time PCR Assay for Hepatitis B Virus DNA Quantification▿

Hepatitis B virus (HBV) DNA quantification is used to establish the prognosis of chronic HBV-related liver disease, to identify those patients who need to be treated, and to monitor the virologic response and resistance to antiviral therapies. Real-time PCR-based assays are gradually replacing other technologies for routine quantification of HBV DNA in clinical practice. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of the real-time PCR Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) platform for HBV DNA quantification. Specificity was satisfactory (95% confidence interval, 98.1 to 100%). Intra-assay coefficients of variation ranged from 0.22% to 2.68%, and interassay coefficients of variation ranged from 1.31% to 4.13%. Quantification was linear over the full dynamic range of quantification of the assay (1.7 to 8.0 log10 IU/ml) and was not affected by dilution. The assay was accurate regardless of the HBV genotype. Samples containing HBV DNA levels above 4.5 log10 IU/ml were slightly underestimated relative to another accurate assay based on branched-DNA technology, but this is unlikely to have noteworthy clinical implications. Thus, the CAP/CTM HBV DNA assay is sensitive, specific, and reproducible, and it accurately quantifies HBV DNA levels in patients chronically infected by HBV genotypes A to F. Samples with HBV DNA concentrations above the upper limit of quantification need to be diluted and then retested. Broad use of fully automated real-time PCR assays should improve the management of patients with chronic HBV infection

Performance of Version 2.0 of the Cobas AmpliPrep/Cobas TaqMan Real-Time PCR Assay for Hepatitis B Virus DNA Quantification ▿

The detection and quantification of hepatitis B virus (HBV) DNA are essential for the diagnosis and treatment of chronic HBV infection. The use of real-time PCR assays for HBV DNA quantification is strongly recommended. The goal of this study was to evaluate the intrinsic characteristics and clinical performance of version 2.0 (v2.0) of the Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) assay, a fully automated platform for HBV DNA quantification in serum or in plasma with a claimed lower limit of detection of 20 IU/ml and a claimed upper limit of quantification of 1.7 × 108 IU/ml. The specificity of the assay was 99% (95% confidence interval, 94.7 to 100%). Intra-assay and interassay coefficients of variation ranged from 0.21% to 2.67% and from 0.65% to 2.25%, respectively. The calibration of the assay was found to be satisfactory. Study of blood specimens from patients infected with HBV genotypes A to F showed good correspondence between HBV DNA levels measured by the CAP/CTM v2.0 assay, version 1.0 of the same assay, and the third-generation “branched DNA” assay. The CAP/CTM v2.0 assay quantified HBV DNA levels in serum or plasma from the same patients equally. In conclusion, the new version of the CAP/CTM assay is sensitive, specific, and reproducible. It accurately quantifies HBV DNA levels in patients chronically infected with HBV genotypes A to F. Improvements made to ensure equal quantification of HBV DNA in serum and plasma have been successful. Overall, the CAP/CTM assay, version 2.0, is well suited to monitoring clinical HBV DNA levels according to current clinical practice guidelines

Alignment of the 5′NCR sequences from the subtype 1b strains that were incorrectly classified by Trugene HCV Genotyping Kit and/or INNO-LiPA HCV 1.0 relative to the consensus sequences of the correctly classified strains, including subtype 1a clade I, subtype 1a clade II and subtype 1b.

<p>Positions 107, 204 and 243, that differentiate subtypes 1a and 1b are in bold. The dotted squares represent the location of the INNO-LiPA HCV 1.0 oligonucleotide probes. The result given by each assay is shown on the right.</p

Ability of the different molecular methods tested in this study to correctly identify HCV subtypes 1a and 1b in a series of 500 patients infected by one or the other of these subtypes.

<p>Correct identification with the different techniques tested is shown for all samples, and for samples that could be amplified by PCR in the assay.</p>*<p>The correct HCV genotype 1 subtype was identified by means of direct sequence analysis of a portion of the NS5B gene followed by phylogenetic analysis, the reference method.</p>**<p>In one 1a case and two 1b cases, not enough serum volume was available for testing in the Abbott RealTi<i>m</i>e HCV Genotype II assay.</p