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Assessment of Hepatitis C Viremia Using Molecular Amplification Technologies: Correlations and Clinical Implications

David R. Gretch, MD, PhD; Corazon dela Rosa, MT; Robert L. Carithers Jr., MD; Richard A. Willson, MD, MD; Barbara Williams, PhD; and Lawrence Corey, MD
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From the University of Washington Medical Center and Harborview Medical Center, Seattle, Washington. Acknowledgments: The authors thank Dr. James Perkins of the University of Washington Medical Center, Dr. Merlin Sayers of Puget Sound Blood Center, Dr. Michael Busch of Irwin Memorial Blood Centers, and Dr. Christopher Blagg of the Northwest Kidney Foundation for ongoing collaboration and for providing some of the clinical specimens used in the current study, and Ms. Willa Lee and Ms. Minjun Chung for technical assistance. Requests for Reprints: David Gretch, MD, PhD, Virology Division, 9th Floor, Pacific Medical Center, 1200 12th Avenue South, Seattle, WA 98144. Current Author Addresses: Drs. Gretch, Williams, and Corey: Virology Division, 9th Floor, Pacific Medical Center, 1200 12th Avenue South, Seattle, WA 98144.

Copyright ©2004 by the American College of Physicians

Ann Intern Med. 1995;123(5):321-329. doi:10.7326/0003-4819-123-5-199509010-00001
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Objective: To compare two recently developed molecular techniques for quantitating the levels of hepatitis C virus (HCV) RNA in the serum of patients with a wide spectrum of chronic hepatitis C.

Design: Serum samples from 299 patients with HCV viremia, 101 control patients without HCV infection, and 19 consecutive patients receiving systemic interferon therapy were evaluated by a commercially available branched-chain DNA (bDNA) assay and a quantitative competitive polymerase chain reaction (PCR).

Setting: University-based hepatology clinics and reference virology laboratory.

Patients: Patients with HCV viremia as defined by results of qualitative RNA PCR, including 53 HCV-infected blood donors, 34 patients receiving renal dialysis, and 212 patients attending a hepatology clinic.

Results: Results of in vitro and in vivo experiments indicated that the sensitivity and dynamic range of the PCR assays were greater than those of the bDNA assay. Detection of HCV viremia by the bDNA assay was highly dependent on viral RNA titers, with a sensitivity of 5% at HCV RNA titers of 5.0 logs per mL or less and 94% at titers of 5.5 logs per mL or greater. The best correlation between assays was observed in specimens with HCV RNA titers between 6.0 and 7.5 logs per mL (r = 0.73). In patients with high-titer HCV viremia, including liver transplant recipients and patients with cirrhosis, quantitative PCR results were an average of 12-fold higher than bDNA assay results. Results of repetitive testing of discordant specimens showed that these discrepancies were caused by a high kit-to-kit coefficient of variation (112%) in the bDNA assay. Of 19 patients receiving interferon therapy, 9 (47%) became bDNA negative, but only 5 became quantitative PCR negative. The bDNA-negative, quantitative PCR-positive patients all had relapse when therapy was discontinued.

Conclusions: The bDNA assay has a narrower linear range for quantitation of HCV viremia than quantitative PCR. Because persons with low HCV titers may respond well to therapy, seropositive persons with negative bDNA results should be retested with PCR-based assays. Similarly, the bDNA assay may underestimate the true degree of HCV viremia in persons with endstage infection (> 107 RNA equivalents/mL of sera). Despite these limitations, the combination of bDNA- and PCR-based assays appears to be optimal for selecting and following patients during interferon therapy.


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Figure 1.
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 circles

A 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).

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Figure 4.
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.

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Figure 3.
Variation in branched-DNA (bDNA) assay results on repetitive testing of specimens obtained from liver transplant recipients and patients with cirrhosis.nnnn

Hepatitis 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).

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Figure 2.
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.

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