Test ID: HCVQU
Hepatitis C Virus (HCV) RNA Detection and Quantification by Real-Time Reverse Transcription-PCR (RT-PCR), Serum

Detection of acute hepatitis C virus (HCV) infection (ie, <2 months from exposure) 

Detection and confirmation of chronic HCV infection 

Quantification of HCV RNA in serum of patients with chronic HCV infection (previously anti-HCV antibody-positive) 

Monitoring disease progression in chronic HCV infection and/or response to anti-HCV therapy

The following algorithms are available in Special Instructions:
-Testing Algorithm for the Diagnosis of Hepatitis C
-Chronic Hepatitis C Standard-of-Care Treatment Algorithm: Combined Pegylated Interferon and Ribavirin Therapy
-Chronic Hepatitis C Treatment Algorithm: HCV Genotype 1 with Telaprevir-containing Combination Therapy

-Chronic Hepatitis C Treatment and Monitoring Algorithm: Boceprevir-Containing Combination Therapy (HCV Genotype 1 only)

• Chronic Hepatitis C Standard-of-Care Treatment Algorithm: Combined Pegylated Interferon and Ribavirin Therapy
• Chronic Hepatitis C Treatment Algorithm: HCV Genotype 1 with Telaprevir-Containing Combination Therapy
• Chronic Hepatitis C Treatment and Monitoring Algorithm: Boceprevir-Containing Combination Therapy (HCV Genotype 1 only)
• Testing Algorithm for the Diagnosis of Hepatitis C

Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

Collection Container/Tube: Serum gel

Submission Container/Tube: Polypropylene vial (Supply T465)

Specimen Volume: 2.2 mL

Collection Instructions:

1. Spin down and separate serum from cells within 6 hours of collection.

2. Freeze serum immediately, and ship specimen frozen on dry ice only.

3. If shipment will be delayed for >24 hours, freeze serum at -70 degrees C (up to 35 days) until shipment on dry ice.

Forms: If not ordering electronically, submit a General Request Form (Supply T239) with the specimen.

Immunocompetent individuals infected with hepatitis C virus (HCV) will have anti-HCV antibodies detectable in their serum. In general, 70% to 80% of these individuals will develop chronic infection with ongoing viral replication in the liver and detectable HCV RNA in serum, eventually causing fibrosis and cirrhosis. The other 20% to 30% of infected individuals recover from the infection without evidence of viral replication or the presence of detectable HCV RNA.

In patients with chronic HCV infection, the response to combined interferon-alpha and ribavirin therapy is correlated with pretreatment serum HCV RNA levels (viral load) and HCV genotype. Similarly, the optimal duration of combined interferon and ribavirin therapy can be determined from the patient's pretreatment viral load and HCV genotype. Clinical studies also indicate that a decrease in HCV RNA levels of more than 2 log  IU/mL at 4 weeks and/or 12 weeks of therapy is predictive of an increased chance of achieving a sustained virologic response (undetectable HCV RNA levels in serum 6 months after completing antiviral therapy). Despite typically undergoing a longer duration of treatment (48 weeks versus 24 weeks), patients with chronic infection due to HCV genotypes 1 and 4 generally have less favorable sustained virologic response rates (40%-60%) than those infected with genotypes 2 and 3 (>80%). To determine the duration of treatment and monitor the response to anti-HCV therapy, HCV RNA levels in serum are typically measured in patients at 0 (baseline) 4 weeks (rapid virologic response, RVR), 8 weeks, 12 weeks (early virologic response: EVR), end-of-treatment (24, 28, or 48 weeks depending on HCV genotype and treatment response), and 24 weeks post-treatment (sustained virologic response: SVR).

The following algorithms are available in Special Instructions:
-Testing Algorithm for the Diagnosis of Hepatitis C
-Chronic Hepatitis C Standard-of-Care Treatment Algorithm: Combined Pegylated Interferon and Ribavirin Therapy
-Chronic Hepatitis C Treatment Algorithm: HCV Genotype 1 with Telaprevir-containing Combination Therapy

-Chronic Hepatitis C Treatment and Monitoring Algorithm: Boceprevir-Containing Combination Therapy (HCV Genotype 1 only)

Undetected

This assay has a serum hepatitis C virus (HCV) RNA quantification result range from 43 to 69,000,000 IU/mL (1.63-7.84 log IU/mL).

An "Undetected" result indicates that HCV RNA was not detected in the specimen. A titer result indicates the presence of HCV infection with active viral replication.

A "Detected" result with the comment "HCV RNA level is <43 IU/mL (<1.63 log IU/mL). This assay cannot accurately quantify HCV RNA below this level" indicates that the HCV RNA level is below the lower limit of quantification for this assay. When clinically indicated, follow-up testing by this assay is recommended in 1 to 2 months. For the purposes of assessing response-guided therapy eligibility, an "Undetected" result is required; a "Detected" result below the limit of quantification should not be considered equivalent to an "Undetected" result.

A "Quantitative" result expressed in IU/mL and log IU/mL indicates the degree of active HCV viral replication in the patient. Monitoring HCV RNA levels over time can be important for assessing disease progression or monitoring a patient's response to anti-HCV therapy.

A "Detected" result with the comment "HCV RNA level is >69,000,000 IU/mL (>7.84 log IU/mL). This assay cannot accurately quantify HCV RNA above this level" indicates that the HCV RNA level is above the upper limit of quantification for this assay.

An "Indeterminate" result with the comment "Inconclusive result. Submit a new specimen for testing if clinically indicated" indicates that inhibitory substances may be present in the specimen. When clinically indicated, collection and testing of a new specimen is recommended.

This test is not licensed by the FDA as a screening test for hepatitis C virus (HCV) infections or a diagnostic test to confirm the presence of HCV infection.

Except for immunocompromised patients or patients with suspected acute hepatitis, laboratory evaluation of HCV infection status should begin with HCV serologic testing, including testing for the presence of anti-HCV antibodies (see Recommended Approach to the Diagnosis, Monitoring, and Treatment of Patients with Hepatitis C Virus in Special Instructions). A diagnosis of chronic HCV infection should not be based solely on the presence of detectable or quantifiable HCV RNA in a single serum specimen.

An "Undetected" HCV RNA test result in conjunction with a positive anti-HCV antibody status (assay signal-to-cutoff ratio of > or =3.8 by EIA, or > or =8.0 by chemiluminescence immunoassay: CIA]) does not exclude the possibility of a resolved HCV infection. When clinically indicated, to distinguish between past/resolved HCV infection and chronic HCV infection with episodic viral replication, patients should be retested for HCV RNA in 1 to 2 months.

The presence of anti-HCV antibodies (assay signal-to-cutoff ratio of <3.8 by EIA or <8.0 by CIA) in patients without detectable HCV RNA may be confirmed by RIBA/80181 Hepatitis C Virus Antibody Confirmation by Recombinant Immunoblot Assay (RIBA), Serum.

Quantitative HCV RNA results generated by this assay may be more than 0.5 log IU/mL lower than those of the VERSANT HCV RNA 3.0 Assay (bDNA) among some clinical serum specimens. Patient care providers are encouraged to use the same HCV RNA quantification assay for serial monitoring of HCV RNA levels in individual patients.

According to the manufacturer, HCV RNA levels from some patients with HCV genotype 4 may be underquantified with this assay by up to 1.0 to 1.5 log IU/mL in comparison to other methods. An internal study has confirmed this observation (see Supportive Data).

This assay was confirmed to have a lower limit of quantification of 43 IU/mL based on probit analysis (95% detection rate), with acceptable correlation to expected results and acceptable linearity over the quantification range of the assay. Accuracy of results among hepatitis C virus (HCV) genotypes 1 to 6 was also verified.

The quantification range of this assay is 43 to 69,000,000 IU/mL (1.63-7.84 log IU/mL).

Based on the product insert of this COBAS AmpliPrep/COBAS TaqMan HCV Test, v1.0 (Roche Molecular Systems, Inc., Branchburg, NJ, 10/2008), the overall limit of detection (LoD) for all HCV genotypes was determined to 18 IU/mL, with an LoD of 7 IU/mL for genotype 1 (see table below).

The mean difference in HCV viral load results between the VERSANT HCV RNA 3.0 Assay (bDNA) and this assay (COBAS AmpliPrep/COBAS TaqMan HCV Test) was -0.43 log IU/mL, with 96.7% (29 of 30 specimens with quantifiable results by both assays) of the differences falling within + or - 0.72 log IU/mL of the mean difference and the individual differences ranging from 0.80 to -0.96 log IU/mL. Of note, a supplemental comparison of the 2 assays among 100 unique clinical specimens containing HCV genotype 4 was conducted due to a report of significant discordance between the viral load results of these assays with several HCV genotype 4 strains.(2) Results of this study confirmed the reported discordance between the viral load results of these assays (generally lower viral load results by the COBAS AmpliPrep/COBAS TaqMan HCV Test), with a mean difference in viral load of -0.44 log IU/mL and individual differences ranging from 0.10 to -1.57 log IU/mL.

1. Alter MJ, Kuhnert WL, Finelli L: Centers for Disease Control and Prevention: guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. MMWR Morb Mortal Wkly Rep 2003;52 (No. RR-3):1-14

2. Chevaliez S, Bouvier-Alias M, Brillet R, Pawlotsky JM: Overestimation and underestimation of hepatitis C virus RNA levels in a widely used real-time polymerase chain reaction-based method. Hepatology 2007;46:22-31

3. Ghany MG, Strader DB, Thomas DL, Seeff LB: Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009;49:1335-1374

4. de Leuw P, Sarrazin C, Zeuzem S: How to use virological tools for the optimal management of chronic hepatitis C. Liver Int 2011;31 supp 1:3-12

5. Poordad F: Big changes are coming in hepatitis C. Curr Gastroenterol Rep 2011;13:72-77

The COBAS AmpliPrep/COBAS TaqMan hepatitis C virus (HCV) test is an FDA-approved in vitro nucleic acid amplification test for the quantification of HCV DNA in human serum using the COBAS AmpliPrep instrument for automated viral nucleic acid extraction (generic silica-based capture technique) and the COBAS TaqMan analyzer for automated amplification and detection of  the viral nucleic acid target. This assay targets the highly conserved 5' con-coding region of the HCV genome and generates amplification products that are detected real-time by a sequence-specific TaqMan probe during amplification. The probe contains a reporter fluorophore and a quencher dye that absorbs light emitted by the reporter. Cleavage of the probe physically separates the quencher from the reporter, enabling light emitted by the latter to be detected by a photomultiplier tube. Because amplification and detection are performed simultaneously, amplification products are measured during the exponential phase of DNA amplification regardless of the initial target concentration.(Package insert: COBAS AmpliPrep/COBAS TaqMan HCV Test; Roche Molecular Systems, Inc., Branchburg, NJ, 10/2008)

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