1. Date of download: 6/3/2016 From: Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications Ann Intern Med. 1995;123(5):321-329. doi:10.7326/0003-4819-123-5-199509010-00001 Analysis of hepatitis C virus (HCV) RNA in serial dilutions of high-titer patient serum by two different quantitative assays, branched-DNA (bDNA) and quantitative polymerase chain reaction (Q-PCR), and a highly sensitive qualitative assay for HCV RNA, reverse transcription PCR (RT-PCR).open circlesclosed circlesA single specimen from an immunosuppressed patient with high-titer HCV viremia (9.5 log-transformed RNA molecules per mL by quantitative PCR assay) was subjected to 18 serial 0.5-log dilutions in uninfected human serum. The serial dilutions were analyzed in duplicate by bDNA assay, quantitative PCR, and qualitative PCR. Quantitative data are expressed as log-transformed equivalents per mL for the bDNA assay ( ) and as log-transformed molecules per mL for the quantitative PCR assay ( ). Qualitative results of the bDNA assay and reverse transcription PCR assay are expressed below the graph as + (positive) or − (negative) for each individual dilution tube. The lower cutoff of the bDNA assay was 350 000 equivalents per mL, or approximately 5.5 log- transformed equivalents per mL, as stated by the manufacturer. By comparison, the PCR lower cutoff was 10 000-fold lower (1.5 log equivalents per mL). Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians

2. Date of download: 6/3/2016 From: Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications Ann Intern Med. 1995;123(5):321-329. doi:10.7326/0003-4819-123-5-199509010-00001 Hepatitis C virus (HCV) RNA titers determined by branched-DNA (bDNA) assay compared with quantitative polymerase chain reaction (Q-PCR) for the 223 total specimens (top) and the 31 specimens from patients with end-stage liver disease (bottom) described inTable 3P( ). The HCV RNA levels are expressed as log-transformed equivalents per mL (log eq/mL) for the bDNA assay on the x-axis and as log-transformed molecules per mL (log molecules/mL) for the quantitative PCR assay on the y-axis. In the top panel, correlation between the two assays was highly significant ( < 0.0001 for the combined patient groups), but the relation appeared nonlinear at the upper and lower ranges defined by the quantitative PCR assay. The specimens from liver transplant recipients showed greater discrepancy in HCV RNA titers determined by the two assays. Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians

3. Date of download: 6/3/2016 From: Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications Ann Intern Med. 1995;123(5):321-329. doi:10.7326/0003-4819-123-5-199509010-00001 Variation in branched-DNA (bDNA) assay results on repetitive testing of specimens obtained from liver transplant recipients and patients with cirrhosis.nnnnHepatitis C virus (HCV) RNA titers were determined by the bDNA assay before dilution (data set A) and after 10-fold dilution and retesting in a new bDNA kit (data set B). The data are presented as log-transformed equivalents per mL (that is, the final results are corrected for dilution). ○ = specimens from liver transplant recipients ( = 10), ● = specimens from patients with end-stage liver disease ( = 9), and filled diamond equals specimens from patients attending a hepatology clinic for evaluation of chronic hepatitis C ( = 6) or from patients receiving renal dialysis ( = 4). Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians

4. Date of download: 6/3/2016 From: Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications Ann Intern Med. 1995;123(5):321-329. doi:10.7326/0003-4819-123-5-199509010-00001 Monitoring HCV RNA levels during interferon-α (IFN) therapy by bDNA and quantitative PCR (Q-PCR).Patients were treated with IFN, 3 MU three times per week for 24 weeks; two patients (bottom left and right panels) received additional interferon therapy. The HCV RNA titers were determined by the bDNA and Q-PCR assays on a monthly basis during therapy. Serum alanine aminotransferase (ALT) levels are shown in each panel (reference range, 6 to 43 IU/L). The patient described in the top left panel was classified as having no virologic response at the end of therapy, although a partial response was evident at 2 months. The patient described in the top right panel was classified as having a virologic response: Results of both HCV RNA assays became negative after the first 2 weeks of therapy, and the patient has remained HCV RNA negative with normal ALT levels during the 8-month post-therapy follow-up. The bottom left panel shows discordance between the bDNA and Q-PCR assays: The patient had a rapid antiviral response by bDNA (negative by 1 month) but a slow response by Q-PCR (positive until month 5). After stopping therapy at 24 weeks, the patient showed a rapid virologic relapse by Q-PCR (positive at month 6 [week 26]), whereas the relapse was not detected by bDNA until month 8. After 14 months without therapy, this patient was retreated with an increased IFN dose (5 MU three times per week for 24 weeks) and had a complete virologic and biochemical response (HCV RNA negative and normal ALT values). The bottom right panel describes a patient who appeared to have a complete virologic response to an escalated dose of IFN by bDNA assay and by serum ALT values but had only a partial response by Q-PCR assay. This patient became transiently negative for HCV RNA by bDNA in month 4 and became persistently negative by bDNA between months 8 and 11. During this period, serum ALT values returned to normal. However, the patient was continuously viremic by the more sensitive Q-PCR assay. Interferon therapy was discontinued before month 11 after ALT values had been normal for 5 months. The patient had a clinical relapse (abnormal ALT values) within 1 month stopping of therapy. RT-PCR equals reverse transcription polymerase chain reaction. To convert the ALT values to µkat/L, multiply by 0.01667. Figure Legend: Copyright © American College of Physicians. All rights reserved.American College of Physicians