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Background: Molecular diagnostic tests for HBV, HCV and HIV-1 and other pathogens are widely used for clinical management. Practical issues related to workflow and labor requirements need to be cha...

BACKGROUND Diagnosis of HCV, HBV, and HIV involves antibody screening followed by confirmation and/or treatment decision using nucleic acid tests. However, minimal data exist evaluating the risk of nucleic acid cross-contamination on serology devices upstream of molecular testing despite the potential clinical and laboratory workflow advantages of single specimen vial testing for both procedures. METHODS We conducted a checkerboard study investigating the potential risk of HCV, HBV, and HIV nucleic acid cross-contamination on 480 negative specimens by a serology screening instrument that uses disposable tips for sample transfer, rather than a fixed needle, before molecular testing. RESULTS Nucleic acid contamination was observed in 0 of 480 negative specimens when processed with alternating high-titer HCV, HBV, or HIV specimens on the serology platform. CONCLUSIONS This study suggests that specimens analyzed by a serology instrument using disposable tips for sample transfer may be suitable for direct primary specimen reflex testing by a sensitive nucleic acid confirmatory test.

Background HIV-1 viral load assays are essential tools for clinical management of people living with HIV-1. Objectives and study design We evaluated concordance between three assays: the cobas® HIV-1 test for use on the cobas® 6800 and cobas® 8800 systems (cobas HIV-1); the COBAS® TaqMan® HIV-1 Test, v2.0 for use with the High Pure System and the COBAS® AmpliPrep/COBAS® TaqMan® HIV-1 test, v2.0. Analytical sensitivity, precision and accuracy of all three methods were assessed using the WHO 2nd International Standard for HIV-1, with concentrations from 5 to 1000 copies/mL. Accuracy and concordance were evaluated using 212 clinical specimens. Overall percent agreement (OPA) was determined using three different thresholds used as medical decision points. Results The limit of detection was below 20 copies/mL for each assay. The hit rate for each assay was 100 % for concentrations ≥ 50 copies/mL. Only the cobas HIV-1 test generated quantifiable data for all replicates at 50 copies/mL. Between 50 and 400 copies/mL, results for all assays were accurate within 0.09 log10 copies/mL, with standard deviation less than 0.14 log10 copies/mL. The mean difference between paired results in clinical specimens ranged from −0.050 to 0.107 log10 copies/mL across all assay comparisons. The OPA between pairs of assays ranged from 94.8 to 98.1% at the 50 copies/mL cutoff, and improved to a range of 97.6–99.0% at the 200 copies/mL cutoff. At the 1000 copies/mL cutoff, OPA between assays was 98.5–99.0%. Conclusions The cobas HIV-1 assay is highly sensitive, accurate and suitable for use in clinical practice.

Background and objectives Accurate, sensitive, and specific tests for detection and monitoring of hepatitis C virus (HCV) RNA concentrations are essential for diagnosis and management of HCV infections. We evaluated the next-generation reverse-transcription real-time PCR test, cobas® HCV test for use with the cobas® 6800/8800 systems (“cobas HCV”) by determining its analytical performance characteristics and clinical utility for the diagnosis and therapeutic monitoring of chronic HCV infections. Methods The limit of detection (LOD), linearity, precision, specificity, matrix equivalence of plasma and serum, and quantitative agreement with the COBAS® AmpliPrep/COBAS® TaqMan® HCV Test version 2.0 (“CAP/CTM HCV v2”) were evaluated. Clinical utility for the diagnosis of chronic HCV infection was demonstrated by testing plasma from HCV seropositive individuals and comparing results to a nucleic acid amplification test (NAAT) approved for use in the diagnosis of chronic hepatitis C. Clinical specificity was investigated by testing plasma from HCV antibody negative subjects with non-HCV related liver diseases. Utility for monitoring treatment response was defined by testing plasma collected during treatment of HCV genotypes (GT) 1, 2, and 3 and determining positive predictive value (PPV), negative predictive value (NPV) and the odds ratio (OR) for predicting cure (sustained virologic response 12 weeks after treatment cessation, “SVR12”). Results The cobas HCV test demonstrated an LOD of at least 15 IU/mL and measurable range from 15 to at least 1.0E + 08 IU/mL (1.2–8.0 log10 IU/mL) for GT 1–6, with high accuracy (≤0.16 log10 difference) and precision (standard deviation 0.04–0.14 log10) throughout the linear range. Paired plasma and serum samples showed highly correlated performance (R2 = 0.97). Quantification was 100% specific for HCV in analytical studies. Correlation with CAP/CTM HCV v2 was high in patient samples (mean titer difference: 0.05 log10 with a 95% CI: 0.03–0.06 log10). For the diagnosis of chronic HCV, positive and negative percent agreement between cobas HCV and the comparator NAAT were 98.8–100% on the cobas 6800 and 8800 systems. Clinical specificity of cobas HCV using samples from HCV antibody negative subjects with non-HCV related liver diseases was 99.6% and 100% on cobas 6800 and 8800 systems. In therapeutic monitoring and SVR12 prediction during experimental treatment for chronic HCV GT 1 infections, undetectable HCV RNA by cobas HCV at different on-treatment weeks had a PPV 76.8%–79.4%, NPV 29.9%–100%, and OR 1.64–47.52. During therapy of HCV GT 2 and GT 3, treatment week 4 and 12 results were: PPV, 84.7% and 75.3%; NPV, 47.8% and 50.0%; OR, 5.09 and 3.05. Conclusions The cobas HCV test is highly sensitive, specific, and accurate HCV RNA test for GT 1–6. It demonstrates excellent correlation with the FDA-approved CAP/CTM HCV v2 test. It is useful clinically for detection of active HCV infection in individuals that have had a positive anti-HCV antibody test result and in monitoring treatment response.

This study evaluated the use of dried blood spot (DBS) for HCV viral load quantification using the COBAS® AmpliPrep/COBAS® Taqman® HCV Quantitative Test v2.0 (CAP/CTM HCV v2), and compared two different procedures for preparation of DBS samples with a Specimen Pre-Extraction (SPEX) reagent (either heated [SPEX with SH] for 10min at 56°C on a thermomixer, or incubated for 1h at room temperature [SPEX at RT]) against the standard plasma input. Whole blood specimens from 48 patients with chronic HCV infection and Whatman® 903 Protein Saver Cards were used to prepare 35μL DBS. An aliquot of plasma was spun and frozen from each draw. Mean DBS viral load results were compared to the corresponding results from plasma. Correlation between DBS to plasma was linear for both SPEX with SH (R2=0.96) and SPEX at RT (R2=0.97) procedures, with a constant negative offset of approximately 2.0log10IU/mL between whole blood DBS without any adjustments and plasma results. After volume corrections, the mean offset to plasma decreased to -0.39 and -0.36 for the two procedures, respectively. The study demonstrated the use of DBS for HCV viral load correlates well with plasma with a constant offset.

BACKGROUND AND OBJECTIVES: Measurement of HIV-1 viral load (VL) is necessary to monitor treatment efficacy in patients receiving antiretroviral therapy. We evaluated the performance of the cobas® HIV-1 quantitative nucleic acid test for use on the cobas® 4800 system ("cobas 4800 HIV-1"). METHODS: Limit of detection, linearity, accuracy, precision, and specificity of cobas 4800 HIV-1, COBAS® AmpliPrep/COBAS® Taqman® HIV-1 version 2.0 (CAP/CTM HIV-1 v2) and Abbott RealTime HIV-1 were determined in one or two out of three sites. RESULTS: The limit of detection of the cobas 4800 HIV-1 for 400 μL and 200 μL input volumes was 14.2 copies/mL (95% CI: 12.5-16.6 copies/mL) and 43.9 copies/mL (37.7-52.7 copies/mL), respectively. Cobas 4800 HIV-1 demonstrated 100% specificity, and results were linear for all analyzed group M HIV-1 subtypes. Precision was high (SD

Accurate, sensitive, and specific tests for detection and monitoring of hepatitis C virus (HCV) RNA concentrations are essential for diagnosis and management of HCV infections. We evaluated the next-generation reverse-transcription real-time PCR test, cobasThe limit of detection (LOD), linearity, precision, specificity, matrix equivalence of plasma and serum, and quantitative agreement with the COBASThe cobas HCV test demonstrated an LOD of at least 15 IU/mL and measurable range from 15 to at least 1.0E + 08 IU/mL (1.2-8.0 logThe cobas HCV test is highly sensitive, specific, and accurate HCV RNA test for GT 1-6. It demonstrates excellent correlation with the FDA-approved CAP/CTM HCV v2 test. It is useful clinically for detection of active HCV infection in individuals that have had a positive anti-HCV antibody test result and in monitoring treatment response.

Background HBV DNA is the most important molecular marker in hepatitis B, used to determine treatment indication and monitoring. Most patients require lifelong hepatitis B virus (HBV) management, thus viral load (VL) monitoring may be performed at different laboratories, with different HBV assays, which may result in different VL results. This multicenter study compares the commutability and concordance of results from four different HBV DNA assays: CAP/CTM HBVv2, HPS/CTM HBVv2 and the new cobas 6800/8800 HBV and cobas 4800 HBV assays. Methods Across all four assays, HBV limit of detection (LoD) and linearity at lower concentrations were assessed using panels traceable to the World Health Organization international standard, and concordance was investigated at the important medical decision cutoffs 2000 and 20,000 IU/ml, using specimens from HBV-positive patients. Results The calculated LoD via a probit curve was 2.7 IU/ml for cobas 6800/8800 HBV, 2.8 IU/ml for cobas 4800 HBV, 9.6 IU/ml for CAP/CTM HBVv2, and 6.2 IU/ml for HPS/CTM HBVv2. The average accuracy was comparable between cobas 6800/8800 HBV, cobas 4800 HBV and CAP/CTM HBVv2 (0.04–0.05 log 10 IU/ml), while a slightly lower accuracy was documented for HPS/CTM HBVv2 (−0.16 log 10 IU/ml). A total of 211–245 clinical samples were used for a pairwise comparison. Mean paired log differences ranged from −0.17 log 10 IU/ml to −0.01 log 10 IU/ml. Coefficient of determination was over 98% for all pairs with high overall percent agreement at the 2000 and 20,000 IU/ml cutoffs (from 91.7% to 96.3%). In a subset of samples with VL±0.5 log 10 to the 2000 and 20,000 IU/ml thresholds, concordance was still 72% and 82%, respectively. Conclusions The new cobas 6800/8800 HBV and 4800 HBV assays show high accuracy in samples with low-level viremia and a high concordance with the established HBV tests, CAP/CTM HBVv2 and HPS/CTM HBVv2, at 2000 and 20,000 IU/ml. Thus, all four HBV assays have high commutability and may be used interchangeably in routine clinical practice.

Hepatitis C virus (HCV) intergenotypic recombinant forms have been reported for various HCV genotypes/subtypes in several countries worldwide. In a recent study, four patients living in Austria had been identified to be possibly infected with a recombinant HCV strain. To clarify results and determine the point of recombination, full-genome next-generation sequencing using the Illumina MiSeq v2 300 cycle kit (Illumina, San Diego, CA, USA) was performed in the present study. Samples of all of the patients contained the recombinant HCV strain 2k/1b. The point of recombination was found to be within the HCV NS2 gene between nucleotide positions 3189-3200 based on H77 numbering. While three of four patients were male and had migration background from Chechnya (n = 2) and Azerbaijan (n = 1), the forth patient was a female born in Austria. Three of the four patients including the female had intravenous drug abuse as a risk factor for HCV transmission. While sequencing techniques are limited to a few specialized laboratories, a genotyping assay that uses both ends of the HCV genome should be employed to identify patients infected with a recombinant HCV strain. The correct identification of recombinant strains also has an impact considering the tailored choice of anti-HCV treatment.

The efficacy of antiviral treatment for chronic hepatitis C virus (HCV) infection is determined by measuring HCV RNA at specific time points throughout therapy using highly sensitive and accurate HCV RNA assays. This study compared the performances of two recently developed real-time PCR HCV RNA assays, cobas HCV for use on the cobas 6800/8800 systems (cobas 6800/8800 HCV) and cobas HCV for use on the cobas 4800 system (cobas 4800 HCV), with those of two established assays, the Cobas AmpliPrep/Cobas TaqMan HCV quantitative test, version 2 (CAP/CTM v2) and the Cobas TaqMan HCV test, version 2 for use with the High Pure system (HPS/CTM v2). The limits of detection (LODs) and linearity at lower concentrations (5 to 1000 IU/ml) were assessed for cobas 6800/8800 HCV and cobas 4800 HCV using WHO standard traceable panels representing HCV genotypes (GT) 1 to 4. Pairwise assay comparisons were also performed using 245 clinical samples representing HCV GT 1 to GT 4. Results from cobas 6800/8800 HCV and cobas 4800 HCV were linear at low HCV RNA concentrations (10 IU/ml difference between expected and observed results) with LODs of 8.2 IU/ml and 11.7 IU/ml, respectively, for GT 1. The new assays showed excellent agreement with results from CAP/CTM v2 and HPS/CTM v2 in samples with quantifiable viral loads. The concordances using the 6 million IU/ml cutoff were high among all four assays (90 to 94%). In conclusion, the cobas 6800/8800 HCV and cobas 4800 HCV tests are sensitive and linear and correlate well with the established Roche assays used in clinical practice.