Clinical laboratory evaluation of a new sensitive and specific assay for qualitative detection of hepatitis C virus RNA in clinical specimens

Sawyer, Lynette; Leung, Kenneth; Friesenhahn, Michel; Duey, D.; McMorrow, Martin; Eguchi, Brandon

doi:10.1016/s0168-8278(00)80780-1

Cited by 19 publications

(23 citation statements)

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“…This statistical approach to resolving gray zone specimens is possible because HCV PCR is highly specific (11). Our data demonstrate that the limit of detection of the HCV TMA assay was 6 IU/ml, which is consistent with the 5-IU/ml limit of detection reported by Ross et al (15) and Sawyer and colleagues (19). Published data have shown that HCV TMA is also very specific (15,19).…”

Section: Discussionsupporting

confidence: 92%

“…Our data demonstrate that the limit of detection of the HCV TMA assay was 6 IU/ml, which is consistent with the 5-IU/ml limit of detection reported by Ross et al (15) and Sawyer and colleagues (19). Published data have shown that HCV TMA is also very specific (15,19). HCV TMA reports specimens as being either reactive (HCV RNA detected) or nonreactive (HCV RNA not detected).…”

Section: Discussionsupporting

confidence: 91%

“…The target RNA is then amplified with an isothermal TMA process that requires the addition of primers, reverse transcriptase, and T7 RNA polymerase. Detection of the amplified product is based on hybrid protection and the dual kinetic assays (18,19). Each tube produces a chemiluminescent signal that is read as relative light units (RLU).…”

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confidence: 99%

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Qualitative Detection of Hepatitis C Virus RNA: Comparison of Analytical Sensitivity, Clinical Performance, and Workflow of the Cobas Amplicor HCV Test Version 2.0 and the HCV RNA Transcription-Mediated Amplification Qualitative Assay

et al. 2002

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The qualitative Cobas Amplicor hepatitis C virus (HCV) version 2.0 assay (HCV PCR) and the BayerReference Testing Laboratory HCV RNA transcription-mediated amplification assay (HCV TMA) were compared for analytical sensitivity, clinical performance, and workflow. Limits of detection were determined by testing dilutions of the World Health Organization HCV standard in replicates of 15 at concentrations of from 1.0 to 70 IU/ml. The limit of detection of the HCV PCR assay was calculated to be 45 IU/ml on initial testing and 32 IU/ml after resolution of gray zone results. The calculated limit of detection for HCV TMA was 6 IU/ml. To compare clinical performance, 300 specimens, grouped as follows, were evaluated: 112 samples that were indeterminate in an anti-HCV enzyme immunoassay (EIA) and for which HCV RNA was not detected by HCV PCR; 79 samples that were EIA positive and for which HCV RNA was not detected by HCV PCR; and 105 samples that were both EIA and HCV PCR positive. For these groups, interassay concordance ranged from 96.2% to 100%. In addition, three HCV PCR gray zone specimens and one neonatal specimen were also evaluated. A 64-sample run (full run, 91 specimens) required 5 h for testing by HCV TMA, whereas almost 8 h were required to test a full run of 22 specimens by HCV PCR. HCV TMA demonstrated excellent concordance with HCV PCR when clinical samples were tested. However, HCV TMA was more sensitive than HCV PCR, required less time for test result completion, and had a greater throughput.It is estimated that 170 million people worldwide are chronically infected with hepatitis C virus (HCV), a major cause of cirrhosis and liver cancer (10,20). Given the burden of the disease and the current potential for cure, there is a compelling need for diagnosis of active HCV infection (10,20). Although diagnostic tests that employ enzyme immunoassays (EIA) and recombinant immunoblot assays are used for serological screening and confirmation, respectively, nucleic acid testing and HCV antigen detection methods are required to demonstrate active infection (3, 9). As HCV antigen tests are still under development, qualitative nucleic acid testing is presently the method of choice to confirm active infection and to assess virologic clearance in response to therapy (3, 9, 22). Qualitative nucleic acid tests are also 10-to 100-fold more sensitive than currently available quantitative tests (9).The qualitative Roche Cobas Amplicor HCV version 2.0 assay (HCV PCR), a reverse transcription-PCR assay, has a reported limit of detection of 50 IU/ml (11) and has been used to confirm viremia and to measure treatment response (10). Recent studies reported that another nucleic acid test based on transcription-mediated amplification (TMA), the HCV RNA transcription-mediated amplification qualitative test (HCV TMA), was able to detect HCV RNA in some patients who had no detectable HCV RNA (as determined by the first and second versions of HCV PCR) at the end of treatment and subsequently experienced virologic relapse (4, 18). These ...

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 91%

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Qualitative Detection of Hepatitis C Virus RNA: Comparison of Analytical Sensitivity, Clinical Performance, and Workflow of the Cobas Amplicor HCV Test Version 2.0 and the HCV RNA Transcription-Mediated Amplification Qualitative Assay

et al. 2002

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“…According to the WHO HCV RNA standard, the analytical sensitivities of the TMA assay are 96% at 5 IU/ml and 100% at 10 IU/ml. The sensitivity of the assay is not influenced by the HCV genotype (35). The clinical specificity is Ͼ99.5%.…”

Section: Methodsmentioning

confidence: 86%

Comparison of Qualitative (COBAS AMPLICOR HCV 2.0 versus VERSANT HCV RNA) and Quantitative (COBAS AMPLICOR HCV Monitor 2.0 versus VERSANT HCV RNA 3.0) Assays for Hepatitis C Virus (HCV) RNA Detection and Quantification: Impact on Diagnosis and Treatment of HCV Infections

Desombere¹,

Vlierberghe

Couvent³

et al. 2005

J Clin Microbiol

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Quantitative measurements of serum hepatitis C virus (HCV) RNA are becoming increasingly important in the management of HCV-infected patients. Here we compared two quantitative assays, the COBAS AMPLICOR HCV Monitor 2.0 assay (Roche Diagnostics) and the branched DNA-based VERSANT HCV RNA 3.0 assay (Bayer Diagnostics) for HCV RNA measurement in 344 samples derived from 120 patients with chronic genotype 1 HCV infection. The overall concordance between the results of the two tests was 95%, and the HCV RNA titers within the dynamic ranges of the assays correlated very well (r 2 ‫؍‬ 0.86). Furthermore, both tests performed equally well in determining an early viral response at week 1 or 4 during antiviral therapy. We also compared two qualitative HCV RNA detection assays: the COBAS AMPLICOR HCV 2.0 assay versus the transcription-mediated amplification (TMA)-based VERSANT HCV RNA qualitative assay. Stored samples from sustained responders to interferon-ribavirin therapy were retested by the HCV TMA assay and were found to contain no detectable HCV RNA, demonstrating complete concordance between the results of PCR and TMA. However, HCV RNA was detected by the TMA assay in end-of-treatment (ETR) samples from 33% of patients with relapses who were HCV RNA negative according to the COBAS AMPLICOR assay. This observation suggests that a TMA assay can lead to a more correct definition of the ETR response.Measurement of hepatitis C virus (HCV) RNA levels has become an integral and increasingly important part of the management of patients with chronic HCV infection: (i) demonstration of the presence of HCV RNA (qualitative test) in the serum or plasma of subjects who test positive for anti-HCV antibodies is essential to prove the existence of an ongoing HCV infection; (ii) definition of the genotype of the infecting HCV isolate is needed to select the appropriate treatment schedule and to estimate the treatment outcome; (iii) measurement of the viral load (quantitative test) at the start of therapy and after 12 weeks of treatment is needed to decide about the usefulness of further treatment (stopping rule); and finally, (iv) demonstration of the presence or absence of HCV RNA (qualitative test) at the end of treatment (ETR) or at the end of follow-up (EFU), 6 or more months later, discriminates sustained responders (SRs) from relapsers (RELs) (3, 7, 8, 12, 17, 19, 21-23, 25, 30, 42, 43).Hepatitis C viral genomes generally appear in the body fluids of infected patients in numbers too low to be detected by simple and direct molecular hybridization-based techniques. Their detection and quantification require strong "amplification" methods that can be introduced as "target amplification," as applied in PCR and transcription-mediated amplification (TMA), or as "signal amplification," as applied in the branched DNA (bDNA) method. The assays most commonly used to quantify HCV RNA are based on the PCR or bDNA technique (3,8,23), methods that differ in analytical sensitivities and dynamic ranges. Whereas the former techniques are ve...

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“…Transcription-mediated amplification (TMA) is an isothermal, autocatalytic target amplification method, which has the potential to detect less than 50 HCV RNA copies/ml (Ͻ10 IU/ml) (21). The high sensitivity of the TMAbased assay may allow for the detection of low HCV RNA levels in end-of-treatment specimens from patients with subsequent virologic relapse.…”

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confidence: 99%

Assessment, by Transcription-Mediated Amplification, of Virologic Response in Patients with Chronic Hepatitis C Virus Treated with Peginterferon α-2a

et al. 2001

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Transcription-mediated amplification (TMA) is an isothermal, autocatalytic target amplification method which has the potential to detect less than 50 hepatitis C virus (HCV) RNA copies/ml (10 IU/ml). The TMA assay was used to assess the presence of residual HCV RNA in plasma from patients treated with polyethylene glycol-modified interferon ␣-2a (peginterferon ␣-2a) who showed a virologic relapse after the end of therapy. Chronic hepatitis C virus (HCV) infection is one of the major causes of the development of cirrhosis and hepatocellular carcinoma (3, 18). The current standard treatment of HCV infection with alpha interferon (IFN-␣) in combination with ribavirin leads to virologic end-of-treatment response in approximately 50% of cases. At the end of a 24-week follow-up period after completion of therapy, approximately 40% of patients achieve a sustained virologic response (13, 17). Thus, in about one of five patients who achieve an end-of-treatment response, a virologic relapse occurs. Rates of virologic relapse are even higher for IFN-␣ monotherapy than for combination therapy with IFN-␣ plus ribavirin. In patients treated with IFN-␣ alone, HCV RNA cannot be detected by reverse transcription-PCR (RT-PCR) at the end of treatment and the end of follow-up in approximately 30 and 15% of cases, respectively (4,6,7,11,12). Thus, ribavirin in the current standard combination treatment enhances virologic end-of-treatment response and reduces relapse rates (13, 16). Recently, treatment of patients with chronic hepatitis C with long-acting polyethylene glycol-modified IFN (peginterferon) resulted in an increase in virologic response rates (24,25). In patients treated for 48 weeks with 180 g of peginterferon ␣-2a, HCV RNA was undetectable by RT-PCR in 69 and 39% of patients at the end of treatment and the end of a 24-week follow-up period, respectively (25).Determination of virologic response is currently based on RT-PCR methods for measurement of HCV RNA in serum or plasma with a lower detection limit of about 100 HCV RNA copies/ml (ϳ50 IU/ml). Transcription-mediated amplification (TMA) is an isothermal, autocatalytic target amplification method, which has the potential to detect less than 50 HCV RNA copies/ml (Ͻ10 IU/ml) (21). The high sensitivity of the TMAbased assay may allow for the detection of low HCV RNA levels in end-of-treatment specimens from patients with subsequent virologic relapse. In a recent pilot study, residual HCV RNA was detected by TMA at the end of treatment in 36% of patients with virologic relapse after standard therapy using IFN-␣ with or without ribavirin (20). In the present study, end-of-treatment and end-of-follow-up plasma samples were investigated by the TMA-based assay to test for residual HCV RNA in a large cohort of patients who were chronically infected with HCV and were treated with standard IFN ␣-2a or peginterferon ␣-2a.

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Clinical laboratory evaluation of a new sensitive and specific assay for qualitative detection of hepatitis C virus RNA in clinical specimens

Cited by 19 publications

References 0 publications

Qualitative Detection of Hepatitis C Virus RNA: Comparison of Analytical Sensitivity, Clinical Performance, and Workflow of the Cobas Amplicor HCV Test Version 2.0 and the HCV RNA Transcription-Mediated Amplification Qualitative Assay

Qualitative Detection of Hepatitis C Virus RNA: Comparison of Analytical Sensitivity, Clinical Performance, and Workflow of the Cobas Amplicor HCV Test Version 2.0 and the HCV RNA Transcription-Mediated Amplification Qualitative Assay

Comparison of Qualitative (COBAS AMPLICOR HCV 2.0 versus VERSANT HCV RNA) and Quantitative (COBAS AMPLICOR HCV Monitor 2.0 versus VERSANT HCV RNA 3.0) Assays for Hepatitis C Virus (HCV) RNA Detection and Quantification: Impact on Diagnosis and Treatment of HCV Infections

Assessment, by Transcription-Mediated Amplification, of Virologic Response in Patients with Chronic Hepatitis C Virus Treated with Peginterferon α-2a

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