All patients underwent a complete virological assay for HBV, HCV and anti-Delta (Delta virus antibody). Anti-HCV antibodies were determined by Enzyme-Linked immunosorbent assay (ELISA assay—Ortho Diagnostic Systems, Raritan, NJ, USA). HCV-RNA (Hepatitis C Virus RNA) levels were detected by polymerase chain reaction using HCV-RNA assay AmpliPrep/COBAS TaqMan (Roche Diagnostics Systems, Branchburg, NJ, USA). Serum samples negative for HCV RNA were retested using the Abbot Real Time HCV assay, with a lower limit of quantification and detection of 12 IU/mL HCV genotypes and subtypes were identified [28 (link)]. HCV viral genotypes were determined by restriction analysis of HCV-RNA 5″ UTR [29 (link)]. Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT); gamma Glutamil Tranferase (γGT); total, conjugated and unconjugated bilirubin; and serum proteins analysis, as well as hematochemical measurements and virological analysis have been executed in the laboratory of Cannizzaro hospital with automated and standardized methods.
Ampliprep cobas taqman
Manufactured by Roche
Sourced in Switzerland, Germany
The AmpliPrep/COBAS TaqMan is a fully automated system for nucleic acid extraction and real-time PCR detection. It is designed for high-throughput molecular diagnostic testing.
Automatically generated - may contain errors
Lab products found in correlation
Architect i1000, by Abbott (2 mentions)
Whole blood, by Qiagen (1 mentions)
Anti hbe, by Roche (1 mentions)
Hbsag, by Roche (1 mentions)
Hbeag, by Roche (1 mentions)
Anti hbs, by Roche (1 mentions)
Anti hbc, by Roche (1 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Ampliprep cobas taqman hiv 1 test, by Roche (1 mentions)
Axsym system, by Abbott (1 mentions)
Chiron riba hcv 3.0 sia, by Ortho Clinical Diagnostics (1 mentions)
Ficoll paque density gradient centrifugation, by Eurobio Scientific (1 mentions)
Facscalibur system, by BD (1 mentions)
Ampliprep cobas taqman hiv 1 test v 2, by Roche (1 mentions)
18 protocols using ampliprep cobas taqman
1
Comprehensive Viral Profiling in Patients
2
Comprehensive Viral Load Profiling
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Serologies for CMV and EBV (DiaSorin, Saluggia, Italy), HBV and HCV (Abbott, Chicago, IL, USA), HHV-8 [23 (link)] were analyzed at baseline. The HIV, HBV and HCV viral loads (VL) were analyzed in plasma using AmpliPrep/COBAS TaqMan (Roche Diagnostics, Basel, Switzerland) and the CMV and EBV loads were measured in whole blood (Qiagen, Hilden, Germany). HHV-8 was quantified by RT-PCR [24 (link)]. CA HIV-1 DNA was quantified by ultrasensitive RT-PCR (Biocentric, Bandol, France) [25 (link)].
3
Hepatitis B Infection Profiling
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All subjects who were HBsAg-positive were then tested for Hepatitis e antigen (HBeAg) and anti-HBeAg, anti-HBcAg, and anti-Hepatitis D virus (HDV) antibodies; HBsAg and HBV-DNA were then quantified, and the HBV genotype was assessed. Serum HBV markers were analysed by commercial immunoenzymatic assays (HBsAg, HBeAg, anti-HBe, anti-HBs, and anti-HBc [total and IgM] from Roche Cobas Diagnostics, North Chicago, IL). Plasma HBV-DNA was quantified by real-time polymerase chain reaction (Roche Cobas® Ampliprep/Cobas® TaqMan). Most patients included in the study had a single point observation for HBV infection since they were in temporary migrant shelters and they moved to other locations after the first visit.
A liver biopsy was proposed to all patients who had a CHB E genotype infection to histologically evaluate liver damage and to define intrahepatic virological parameters. Nine patients gave written informed consent to undergo liver biopsy. For these 9 patients we had the opportunity to have a second follow-up and to further study HBV intrahepatic parameters and liver histology.
A liver biopsy was proposed to all patients who had a CHB E genotype infection to histologically evaluate liver damage and to define intrahepatic virological parameters. Nine patients gave written informed consent to undergo liver biopsy. For these 9 patients we had the opportunity to have a second follow-up and to further study HBV intrahepatic parameters and liver histology.
4
Quantification and Sequencing of HBV DNA
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Biochemical and serological markers, and HBV DNA level, of the patients were routinely detected in the Central Clinical Laboratory of Beijing 302 Hospital. HBV DNA level was determined using a real-time quantitative PCR kit (Fosun Pharmaceutical Co., Ltd., Shanghai, China) with a lower limit of detection (LLOD) of 100 IU/ml before April 2012 and 40 IU/ml afterwards. If sufficient amount of serum was available, HBV DNA of interesting samples below the LLOD was further quantitated using AmpliPrep/COBAS TaqMan (Roche Diagnostics) with an LLOD of 20 IU/ml as described previously [34 (link)]. For HBV resistance testing, HBV DNA was extracted from patient serum with DNA out (Tianenze, Beijing, China) and the HBV RT gene was amplified by nested PCR with an LLOD of 10 IU/ml [27 (link)]. The PCR products were directly sequenced for all samples. Clonal sequencing was performed (≥20 clones/sample at each time point) if necessary. Sequencing was performed by a professional company using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
5
Comprehensive Hepatitis B Viral Markers
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HBsAg, anti-HBc, HBeAg, anti-HBe, and anti-HBs were tested by commercial assays (Vitros, Upjohn, Germany). HBV-DNA was quantified by AmpliPrep/COBAS® TaqMan® (Roche Diagnostics GMBH, Mannheim; Germany, range: 15-100 000 000 IU/mL).
6
Quantification of HBV and HIV Markers
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HBV serological markers were measured using immune-enzymatic assays (Roche/Cobas Diagnostics, Rotkreuz, Switzerland). Plasma HBV-DNA was identified using real-time polymerase chain reaction (lower limit of quantification: 20 IU/ml) (Roche/Cobas Ampliprep/Cobas Taqman, Rotkreuz, Switzerland). Plasma HIV-RNA levels were measured using a commercial test, with 20 copies/ml of HIV-RNA defined as the lower limit of quantification (COBAS Ampliprep/Cobas Taqman HIV-1 Test, v2.0).
7
Quantitative PCR for HBV DNA
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For the purposes of this study, all of the patients were re-tested for HBV DNA by means of quantitative real-time PCR using a COBAS AmpliPrep/COBAS TaqMan automated system (HBV Test, Roche Diagnostics).
8
Infant HIV Transmission Factors
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The variables on which data was extracted were infant sex, age (in week), HIV status, feeding practice, mother ART status at delivery, and PMTCT services status provided for infants and mothers. The outcome variable was HIV infection which was confirmed by automated Polymerase Chain Reaction (PCR) using COBAS AmpliPrep/COBAS TaqMan (Roche CAP/CTM) HIV–1 proviral Deoxyribonucleic Acid (DNA) qualitative detection technique [22 ].
9
Longitudinal Biomarker Monitoring in HBV
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All laboratory data during the trial were collected at 12-week intervals via the Central Lab (GC abs, Yongin, Korea). HBV DNA quantification was performed using a COBAS AmpliPrep/TaqMan test (Roche Diagnostics, Indianapolis, IN) with a lower detection limit of 20 IU/mL. Other laboratory methods were followed as previously reported (13 (link)).
Bone mineral density, non-invasive fibrosis test, and resistance surveillance were regularly performed (see Supplementary Material, Supplementary Digital Content 1,http://links.lww.com/AJG/B491 ). Particularly, FIB-4 and aspartate aminotransferase-to-platelet ratio indexes (APRI) were annually estimated to assess liver fibrosis (see Table, Supplementary Digital Content 2 for formula used, http://links.lww.com/AJG/B485 ).
Bone mineral density, non-invasive fibrosis test, and resistance surveillance were regularly performed (see Supplementary Material, Supplementary Digital Content 1,
10
HCV Antibody Screening and Viral Load
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An HCV-antibody chemiluminescent immunoassay (HCV-Ab) (Architect i1000, Abbott Laboratories, Abbott Park, IL) was used to screen patients for HCV. Positive HCV-Ab tests were confirmed by measuring HCV RNA viral load (Cobas AmpliPrep/TaqMan, Roche Diagnostics, Basel, Switzerland). HCV-Ab testing typically yielded results within 1-3 days and viral load testing typically yielded results within 4 days.
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