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Journal of Clinical Microbiology, April 1998, p. 862-865, Vol. 36, No. 4
Servicio de Microbiologia Clinica y
Enfermedades Infecciosas, Hospital General Universitario Gregorio
Maranon, Madrid, Spain1;
Laboratorio di
Patologia Clinica, Ospedale S. Anna, Como,
Italy2;
Laboratoire de Biologie
Moleculaire, Hopital de Meaux, Meaux, France3;
PCR Unit, Roche Diagnostic Systems, Basel,
Switzerland5;
PCR Labor,
Gemeinschaftspraxis Dres Fenner, Hamburg,
Germany6;
Hopital St. Luc, Montreal,
Quebec, Canada4; and
Roche Molecular
Systems, Somerville, New Jersey7
Received 8 September 1997/Returned for modification 19 November
1997/Accepted 6 January 1998
The benefits shown by the recent introduction of PCR for the in
vitro diagnosis of hepatitis C virus (HCV) infection has prompted the
development of standardized, ready-to-use assays that can be
implemented in routine clinical laboratories. We have evaluated the clinical performance of COBAS AMPLICOR HCV
(COBAS), the first instrument system that allows the
automation of HCV RNA amplification and detection, to determine its
performance in the routine laboratory setting. More than 2,000 specimens collected at five centers were analyzed in parallel by the
COBAS and the manual AMPLICOR HCV (AMPLICOR) tests, and the
results were compared with the results for biochemical and serological
markers of HCV. In this study the two PCR systems showed the same
accuracy, with a concordance rate of 99.8%. As expected, the
correlation between serology and PCR was not absolute because the
presence of anti-HCV antibodies may be associated with a latent or past
infection. On the other hand, if the presence of confirmed anti-HCV
antibodies and elevated alanine aminotransferase levels are taken as
the "gold standard," indicating an active, ongoing infection,
the COBAS and AMPLICOR tests show high and comparable
sensitivities (100%) and specificities (98%), with positive and
negative predictive values of 100 and 97%, respectively. During the
study no false-positive reactions were detected. The use of an internal
control allowed the identification of inhibitory substances that
prevented amplification for 0.3 and 0.4% of samples tested by the
COBAS and AMPLICOR tests, respectively. Compared to the manual
system, the COBAS system allowed a significant reduction of
hands-on time and could improve the overall laboratory work flow. In
conclusion, these results support the use of the COBAS and
AMPLICOR tests for the molecular diagnosis of active HCV
infections.
The lack of virus isolation
techniques and antigen detection assays has made nucleic acid-based
amplification the method of choice for the direct identification of
hepatitis C virus (HCV). Indeed, by PCR it is possible to assess the
status of the infection, to detect viral replication in seropositive
patients, and to diagnose the infection in immunocompromised patients
and during the window that precedes seroconversion (2, 3, 15, 18,
21).
The implementation of PCR in routine diagnostic laboratories, however,
requires a level of standardization that was not originally provided by
in-house methods. Additionally, by in-house methods the occurrence of
false-positive results due to amplified product contamination is not
always preventable and false-negative results due to inhibition are not
always easily detectable (5, 17, 19, 20, 24). Also, the
hands-on time required by a manual in-house assay for reagent
preparation as well as the testing of samples from patients limits its
utility in the routine diagnostic laboratory. To address these
technical concerns, a dedicated instrument, the COBAS AMPLICOR
HCV (COBAS) analyzer, that automates amplification and detection
has recently been introduced (6, 10). The system provides amplification and detection with standardized,
ready-to-use reagents based on the reagents formerly developed
for the corresponding AMPLICOR HCV manual format
(AMPLICOR). The amplification reagents include the enzyme
uracil-N-glycosylase (AmpErase) for the prevention of
carryover contamination (12, 22) and an internal
control (IC) for the identification of processed specimens containing substances that may interfere with amplification (16).
The closed system provides additional protection against carryover
contamination. In the present study we evaluated the performance of the
COBAS test with a collection of more than 2,000 clinical specimens
and compared it with that of the corresponding manual AMPLICOR test and the results for other serological and biochemical parameters.
We analyzed 2,292 consecutive serum specimens collected and
separately tested at five centers. The five centers provided results for 484, 655, 495, 498, and 160 serum specimens, respectively. Serum
was separated from whole blood and was immediately stored at Serology.
All specimens were tested for the presence of
anti-HCV antibodies by enzyme-linked immunosorbent assay (ELISA; Abbott
Diagnostics, North Chicago, Ill.; Ortho Diagnostic Systems, Raritan,
N.J.) and at most centers by RIBA (Chiron Corp., Emeryville, Calif.). This confirmatory assay was performed with 2,056 (89%) samples. In
addition, biochemical markers of liver inflammation and necrosis including aspartate aminotransferase and alanine aminotransferase (ALT)
levels were evaluated.
PCR analysis.
Serum specimens were analyzed by the COBAS
test (Roche Diagnostic Systems, Somerville, N.J.) by following the
manufacturer's instructions. Briefly, 100 µl of serum was incubated
with a lysis buffer, and the RNA was then precipitated with isopropanol
by centrifugation, washed once with ethanol, and resuspended in 1 ml of
specimen diluent. An IC RNA was also introduced into the specimen with
the diluent and served as an amplification control for each
individually processed specimen. The HCV IC consists of a synthetic RNA
transcript with primer binding regions identical to those of the
HCV target sequence, a randomized internal sequence with a length and a
base composition similar to those of the HCV target sequence, and a
unique probe binding region that differentiates the IC from the target
amplicon.
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multicenter Evaluation of the COBAS AMPLICOR HCV Assay, an
Integrated PCR System for Rapid Detection of Hepatitis C Virus RNA
in the Diagnostic Laboratory
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
80°C
in several aliquots to preserve the integrity of the HCV RNA.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The 2,292 consecutive specimens obtained from patients at risk of HCV infection were simultaneously analyzed by the COBAS and the AMPLICOR tests. The concordance between the two systems was 98%, but it rose to 99.8% after repeat testing (Fig. 1). Discrepant results were observed for 4 (0.2%) of all samples.
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Samples presenting negative IC values due to the presence of inhibitory substances that prevented amplification of the IC were resolved by retesting another aliquot of the specimen. After resolution of the results, invalid results were obtained for 0.3 and 0.4% of all specimens analyzed by the COBAS and AMPLICOR tests, respectively. The values obtained prior to repeat testing were 3 and 2% for the COBAS and AMPLICOR tests, respectively.
When compared to the patients' serological status, as assessed by anti-HCV ELISA and RIBA, the results of PCR showed an overall concordance rate of 84% (Fig. 2).
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To better define the relationship between serological and molecular data for our patients, we compared the results obtained with the COBAS and AMPLICOR tests with those of ALT level and RIBA determinations. The combination of the latter two parameters served to identify active HCV infection. By this approach the concordance rate increased to 98% (Fig. 3), indicating that most anti-HCV-positive, PCR-negative discrepant results were due to past or latent infections. Moreover, if PCR results are compared to the active infection status, with use of active infection status as the "gold standard," the COBAS and AMPLICOR tests show sensitivities and specificities of 96 and 100% and of 95 and 100%, respectively. The corresponding positive predictive and negative predictive values were 100 and 98%, respectively, for the COBAS test and 100 and 97%, respectively, for the AMPLICOR test.
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The turnaround time for the complete COBAS test including specimen preparation and the amplification and detection of HCV and IC was 5 h for a batch of 20 specimens. The operator was required to be present for less than 2 h to perform manual steps that included sample and reagent preparation and loading of the amplification tubes in the thermal cyclers and in the detection racks. The overall workload time was about 5 min/specimen, compared to 7 min/specimen for the manual test. By using the parallel-run feature of the instrument, the throughput can be increased to three to four runs per day, including an unattended overnight run.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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HCV infection is a typical example of a disease in which direct detection of the virus is essential for a correct diagnosis. In contrast to the other available in vitro assays, PCR has the potential for high diagnostic value because it offers a definitive identification of HCV. However, the poor reproducibility of in-house-developed assays, shown in previous studies, has indicated the need for standardized and more accurate procedures (5, 24). The recent introduction of an automated instrument for the amplification and detection of HCV RNA has prompted us to evaluate its performance with a large collection of clinical specimens obtained from different institutions. The results of this study have shown that the COBAS test has the same performance as the established manual AMPLICOR test because only 0.2% of all results remained discordant after repeat testing. Initial discrepancies were due mostly to invalid results caused by low values for the IC.
The COBAS and AMPLICOR tests also showed the same sensitivities and specificities compared to those of serology and clinical diagnosis, which were taken as the gold standards. We used clinical diagnosis because serology alone does not necessarily reflect the presence of an active infection but reflects only a previous exposure to the virus (2, 21). On the other hand, the presence of anti-HCV antibodies in conjunction with elevated ALT levels and histological evidence of chronic hepatitis better represent the features of an ongoing, clinically relevant infection (1, 3, 13, 21). In this regard, the samples with discrepant results that were anti-HCV positive and HCV RNA negative were from patients who had normal ALT levels and hence a latent, inactive infection. The few patients (2%) who had a clinical diagnosis of type C hepatitis but who were PCR negative had borderline ALT level elevations and probably had HCV RNA concentrations below the analytical sensitivity of the AMPLICOR system (7).
We did not compare the COBAS test with nested PCR techniques because the manual AMPLICOR test, which served as the reference for the automated system in this study, had been previously extensively analyzed (8, 9, 14, 23).
An interesting feature of the COBAS test is the optional detection of an IC that is coamplified with the target and that serves to identify the possible presence of interfering substances that inhibit DNA polymerization, causing false-negative reactions (16).
Inhibition occurred with 3% of all samples and was readily solved by repeat testing of frozen aliquots. This may indicate that modification or degradation of inhibitory substances occurred during storage or that inhibitors are not equally distributed throughout a sample (16).
On the other hand, the presence of uracil-N-glycosylase (AmpErase), which destroys a dUTP-containing amplicon from previous reactions, and the use of proper precautions taken at the time of specimen preparation prevented false-positive reactions due to carryover contamination or cross contamination from positive to negative samples (11, 12).
In this respect, an additional benefit of the COBAS system is the closed containment in which all amplification and detection steps take place. Amplification products are kept in sealed tubes that are never opened but that are pierced by the transfer tip during the detection part of the procedure. Throughout the duration of the study we assessed the robustness of the instruments and never had failures requiring external assistance. Taken together, these data suggest that the COBAS test may be a suitable system for the routine analysis of samples obtained from anti-HCV-positive individuals.
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FOOTNOTES |
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* Corresponding author. Mailing address: Roche Diagnostic Systems, Bldg. 222/121, 4070 Basel, Switzerland. Phone: 41 61 6873363. Fax: 41 61 6872109. E-mail: Giuseppe.Colucci.GC1{at}Roche.com.
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REFERENCES |
|---|
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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|---|
| 1. | Alberti, A., G. Morsica, L. Chemello, D. Cavalletto, F. Noventa, P. Pontisso, and A. Ruol. 1992. Hepatitis C viraemia and liver disease in symptom-free individuals with anti-HCV. Lancet 340:697-698[Medline]. |
| 2. |
Alter, H. J.
1995.
To C or not to C: these are the questions.
Blood
85:1681-1695 |
| 3. | Bonino, F., M. R. Brunetto, F. Negro, M. Baldi, G. Saracco, M. L. Abate, A. Fabiano, G. Verme, et al. 1993. Hepatitis C virus infection and disease. Diagnostic problems. J. Hepatol. 17(Suppl. 3):78-82. |
| 4. | College of American Pathologists. 1992. Workload recording method and personnel management manual. College of American Pathologists, Northfield, Ill. |
| 5. | Damen, M., H. T. M. Cuypers, H. W. Zaaijer, H. W. Reesink, W. P. Schaasberg, W. H. Gerlich, H. G. M. Niesters, and P. N. Lelie. 1996. International collaborative study on the second EUROHEP HCV-RNA reference panel. J. Virol. Methods 58:175-185[Medline]. |
| 6. |
DiDomenico, N.,
H. Link,
R. Knobel,
T. Caratsch,
W. Weschler,
Z. G. Loewy, and M. Rosenstraus.
1996.
COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine PCR.
Clin. Chem.
42:1915-1923 |
| 7. |
Dusheiko, G. M.,
S. Khakoo,
P. Soni, and L. Grellier.
1996.
A rational approach to the management of hepatitis C infection.
Br. Med. J.
312:357-364 |
| 8. | Gerken, G., P. Pontisso, M. Roggendorf, M. G. Rumi, P. Simmonds, C. Trepo, S. Zeuzem, and G. Colucci. 1996. Clinical evaluation of a single reaction, diagnostic PCR assay for the detection of hepatitis C virus (HCV) RNA. J. Hepatol. 24:33-37[Medline]. |
| 9. | Goergen, B., K. H. Meyer Zum Buschenfelde, and G. Gerken. 1994. Assessment of viral replication for routine diagnosis of HCV infection and for monitoring antiviral treatment using Amplicor HCV detection system. Klin. Lab. 40:1-7. |
| 10. | Jungkind, D., S. DiRienzo, K. G. Beavis, and N. S. Silverman. 1996. Evaluation of automated COBAS AMPLICOR PCR system for detection of several infectious agents and its impact on laboratory management. J. Clin. Microbiol. 34:2778-2783[Abstract]. |
| 11. | Kwok, S., and R. Higuchi. 1989. Avoiding false positive with PCR. Nature 339:237-238[Medline]. |
| 12. | Longo, M. C., M. S. Berninger, and H. L. Hartley. 1990. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene 93:125-128[Medline]. |
| 13. |
Marin, M. G.,
S. Bresciani,
M. Puoti,
A. Rodella,
A. Gussago,
A. Ravaggi,
G. Pizzocolo,
A. Albertini, and E. Cariani.
1996.
Clinical significance of serum hepatitis C virus (HCV) RNA as marker of HCV infection.
J. Clin. Microbiol.
32:3008-3012 |
| 14. | Prati, D., C. Capelli, P. Bosoni, F. Mozzi, A. Zanella, and G. Sirchia. 1994. Determination of hepatitis C virus RNA in the serum by the Amplicor HCV PCR kit. Vox Sang. 67:112-114[Medline]. |
| 15. | Roggendorf, M., M. Lu, H. Meisel, M. Riffelmann, E. Schreier, and S. Viazov. 1996. Rational use of diagnostic tools in hepatitis C. J. Hepatol. 24:26-34[Medline]. |
| 16. | Rosenstraus, M., Z. Wang, S.-Y. Chang, D. DeBoneville, and J. P. Spadoro. An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J. Clin. Microbiol. 36:191-197. |
| 17. |
Rys, P. N., and D. H. Persing.
1993.
Preventing false positives: quantitative evaluation of three protocols for inactivation of polymerase chain reaction amplification products.
J. Clin. Microbiol.
31:2356-2360 |
| 18. |
Tedeschi, V., and L. B. Seeff.
1995.
Diagnostic tests for hepatitis C: where are we now?
Ann. Intern. Med.
123:383-385 |
| 19. | Victor, T., A. Jordaan, R. du Toit, and P. D. Van Helden. 1993. Laboratory experience and guidelines for avoiding false positive polymerase chain reaction results. J. Clin. Chem. Clin. Biochem. 31:531-535. |
| 20. | White, T. J., R. Madej, and D. H. Persing. 1992. The polymerase chain reaction: clinical applications. Adv. Clin. Chem. 29:161-196[Medline]. |
| 21. | Wilber, J. C., and A. Polito. 1995. Serological and virological diagnostic tests for hepatitis C virus infection. Semin. Gastrointestinal Dis. 6:13-19[Medline]. |
| 22. |
Young, K. K. Y.,
R. M. Resnick, and T. W. Myers.
1993.
Detection of hepatitis C virus RNA by a combined reverse transcription-polymerase chain reaction assay.
J. Clin. Microbiol.
31:882-886 |
| 23. | Young, K. Y., J. J. Archer, O. Yokosuka, M. Omata, and R. Resnick. 1995. Detection of hepatitis C virus RNA by a combined reverse transcription PCR assay: comparison with nested amplification and antibody testing. J. Clin. Microbiol. 33:654-657[Abstract]. |
| 24. | Zaaijer, H. J., H. T. Cuypers, and H. W. Reesink. 1993. Reliability of HCV PCR results. Lancet 341:722-724[Medline]. |
Journal of Clinical Microbiology, April 1998, p. 862-865, Vol. 36, No. 4
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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