Qualitative HBsAg was detected using Monolisa® HBsAg ULTRA (Bio-Rad, Evolis Tween Plus, Marnes- la- Coquette, France), a one-step sandwich enzyme immunoassay. Samples reactive for HBsAg were subsequently tested for HBV DNA and HDV serology. All tests were performed according to manufacturer’s instructions. The quantitative measurement of HBV DNA in plasma was done with the COBAS® AmpliPrep/COBAS® TaqMan® HBV Test (Roche Molecular Systems, Inc. Roche Diagnostics GmbH). The limit of detection of this assay was 20 IU/ml. Testing for anti-HDV antibody was performed using ETI-AB-DELTAK-2, an enzyme immune-assay for the qualitative determination of total antibodies to hepatitis delta antigen (anti-HD) (DiaSorin Limited, United Kingdom).
Cobas ampliprep cobas taqman hbv test
Manufactured by Roche
Sourced in United States, Germany, Switzerland
The COBAS AmpliPrep/COBAS TaqMan HBV test is an automated in vitro diagnostic test for the quantitative determination of Hepatitis B Virus (HBV) DNA in human plasma or serum samples. The test utilizes real-time PCR technology to detect and measure the viral load.
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30 protocols using cobas ampliprep cobas taqman hbv test
1
Hepatitis B and Delta Virus Detection
2
HBV Viral Load in HBsAg Positive Mothers
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HBV viral load was tested on HBsAg positive mothers samples collected at delivery, using COBAS® AmpliPrep/COBAS® TaqMan® HBV Test, version 2.0 (Roche, Mannheim, Germany) with a lower LoD of 20 IU/mL.
3
Quantification of HBV markers
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Serum HBV-DNA quantification was performed by quantitative PCR using Cobas Ampliprep/Cobas Taqman HBV test (Roche Diagnostics, Germany). Serum HBsAg was quantified with Elecsys HBsAg II kit/Cobas e411 (Roche Diagnostics, Germany) according to manufacturer’s instructions. Quantitative levels of HBcrAg were determined using the Lumipulse G HBcrAg assay on the LUMIPULSE G1200 Analyzer (Fujirebio Europe, Belgium) after denaturation of proteins by incubation at 60°C for 30 min according to manufacturer’s instructions. Due to the denaturation step, the assay measures simultaneously denatured HBeAg, HBcAg and the pre-Core protein p22cr (aa −28 to aa 150). The assay measurement linear range spans from 3 to 7 logU/ml, hence adequate quantification is achieved at a concentration of 3 log U/ml (defined as Limit of Quantification or LoQ) or above, and patients below were considered negative.
4
Quantitative HBV DNA Measurement
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The HBV DNA (viral load) level was assessed by commercial real-time PCR (COBAS AmpliPrep/COBAS TaqMan HBV test, version 2.0, Roche Diagnostics, Germany) for automated specimen processing and amplification, with a lower limit of detection of approximately 20 IU/mL. If the samples had detectable HBV DNA, a quantitative real-time PCR analysis was performed to determine the level of DNA.
5
Quantitative HBV Biomarker Analysis
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Qualitative HBeAg and Quantitative HBsAg levels were determined by using the Roche Cobas reagent kits (Roche Diagnostics). The HBV DNA viral load (IU/mL) was determined by using Cobas AmpliPrep/Cobas TaqMan HBV Test (Roche Diagnostics).
6
Hepatitis B Virus Reactivation During DAA Therapy
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The definition of HBV co-infection is seropositive HBsAg before DAA treatment. HBV DNA was quantified by The Roche COBAS® AmpliPrep/COBAS® TaqMan® HBV Test, v2.0. The definition of inactive HBV carrier is HBV DNA ≤2000 IU/mL before DAA treatment. HBV reactivation was defined as an HBV DNA increase of 2 log from baseline or rising above 100 IU/mL from undetectable status [11 (link)]. HBV-related hepatitis is defined as viral reactivation with abrupt elevation of serum ALT to >5× upper limit of normal. Advanced fibrosis is defined as fibroscan >9.5 kPa while cirrhosis is defined as fibroscan >12.5 kPa [13 (link),14 (link)]. Pre-DAA liver biopsy was done in only 4 patients. The definition of hepatic decompensation includes jaundice with bilirubin >3.0 mg/dL, coagulopathy with INR >2.0, new diagnosed ascites by liver echo, new esophageal varices requiring endoscopic treatment, or hepatic encephalopathy diagnosed by clinical physicians. Hepatocellular carcinoma was diagnosed by liver dynamic contrast-enhanced computed tomography and/or liver biopsy.
7
Quantitative PCR for HBV Viral Load
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All initial non-discriminated reactive samples were tested for viral load using the fluorescent quantitative PCR method by COBAS® AmpliPrep/COBAS® TaqMan® HBV Test (Roche Molecular Systems, Inc., South Branchburg, NJ, United States). According to the instructions of the reagents, the results were categorized into three types: (i) negative, no HBV-DNA detected; (ii) <12 IU/mL, HBV-DNA detected but below the detection range; and (iii) >12 IU/mL, HBV-DNA detected and above the detection range, with specific values representing the viral load.
8
Quantification of HBV DNA and HBsAg
9
Serological Markers for Hepatitis B
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We evaluated the levels of HBsAg, HBeAg, and HBV DNA in the serum using a commercial blood test. We also analyzed the anti-HBc IgG levels, and checked qualitatively prior to initiating antiviral therapy using Architect Anti-HBc assay (Abbott Laboratories, IL, USA). A indirect relationship was shown between the amount of analyte in the sample and the RLUs detected by Architect System optics. Results were calculated as normalized signal cut-off (S/Co) ratios obtained by measuring the signal strength of sample and the signal strength of an internal cut-off. IgG anti-HBc positivity was defined by an S/Co ratio ≥ 1.0. The indirect ratio of light absorbance of anti-HBc IgG levels was measured quantitatively. In previous report, indirect ratio of anti-HBc IgG was correlated to quantified anti-HBc IgG levels [10 (link)]. The level of HBV DNA in the serum was quantified using a commercially available real-time polymerase chain reaction assay (COBAS AmpliPrep-COBAS TaqMan HBV test, detection limit = 12 IU/mL; Roche Diagnostics, Basel, Switzerland). HBV DNA was routinely estimated at intervals of six months.
10
Quantifying Serum HBV DNA Levels
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Serum HBV DNA levels were quantified using a commercially available real-time polymerase chain reaction assay (COBAS AmpliPrep-COBAS TaqMan HBV test, detection limit = 12 IU/mL; Roche Diagnostics, Basel, Switzerland). Measurements of HBV DNA were routinely performed at every 6 months.
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