Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA

Guido Gerken, Patrizia Pontisso, Michael Roggendorf, Maria Grazia Rumi, Peter Simmonds, Cristian Trepo, Stefan Zeuzem, Giuseppe Colucci

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Background/Aims: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). Methods: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. Results: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV; both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. Conclusions: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

Original languageEnglish
Pages (from-to)33-37
Number of pages5
JournalJournal of Hepatology
Volume24
Issue number1
DOIs
Publication statusPublished - Jan 1 1996

Fingerprint

Hepacivirus
RNA
Polymerase Chain Reaction
Chronic Hepatitis C
Chronic Hepatitis
Autoimmune Hepatitis
DNA-Directed RNA Polymerases
Sensitivity and Specificity
Uracil
Serology
Routine Diagnostic Tests
Reverse Transcription
Renal Dialysis
Genome
Antibodies
Serum

Keywords

  • Chronic hepatitis
  • Clinical samples
  • HCV RNA
  • PCR

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA. / Gerken, Guido; Pontisso, Patrizia; Roggendorf, Michael; Rumi, Maria Grazia; Simmonds, Peter; Trepo, Cristian; Zeuzem, Stefan; Colucci, Giuseppe.

In: Journal of Hepatology, Vol. 24, No. 1, 01.01.1996, p. 33-37.

Research output: Contribution to journalArticle

Gerken, G, Pontisso, P, Roggendorf, M, Rumi, MG, Simmonds, P, Trepo, C, Zeuzem, S & Colucci, G 1996, 'Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA', Journal of Hepatology, vol. 24, no. 1, pp. 33-37. https://doi.org/10.1016/S0168-8278(96)80183-8
Gerken, Guido ; Pontisso, Patrizia ; Roggendorf, Michael ; Rumi, Maria Grazia ; Simmonds, Peter ; Trepo, Cristian ; Zeuzem, Stefan ; Colucci, Giuseppe. / Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA. In: Journal of Hepatology. 1996 ; Vol. 24, No. 1. pp. 33-37.
@article{31852cdfa52a4a9a99a80fc1023cf3ab,
title = "Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA",
abstract = "Background/Aims: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). Methods: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. Results: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100{\%} and 99.3{\%} for the former and of 98.8{\%} and 100{\%} for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64{\%} and 36{\%} of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV; both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. Conclusions: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.",
keywords = "Chronic hepatitis, Clinical samples, HCV RNA, PCR",
author = "Guido Gerken and Patrizia Pontisso and Michael Roggendorf and Rumi, {Maria Grazia} and Peter Simmonds and Cristian Trepo and Stefan Zeuzem and Giuseppe Colucci",
year = "1996",
month = "1",
day = "1",
doi = "10.1016/S0168-8278(96)80183-8",
language = "English",
volume = "24",
pages = "33--37",
journal = "Journal of Hepatology",
issn = "0168-8278",
publisher = "Elsevier B.V.",
number = "1",

}

TY - JOUR

T1 - Clinical evaluation of a single reaction, diagnostic polymerase chain reaction assay for the detection of hepatitis C virus RNA

AU - Gerken, Guido

AU - Pontisso, Patrizia

AU - Roggendorf, Michael

AU - Rumi, Maria Grazia

AU - Simmonds, Peter

AU - Trepo, Cristian

AU - Zeuzem, Stefan

AU - Colucci, Giuseppe

PY - 1996/1/1

Y1 - 1996/1/1

N2 - Background/Aims: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). Methods: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. Results: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV; both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. Conclusions: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

AB - Background/Aims: In the past few years the detection of HCV-RNA by polymerase chain reaction has become a well-established diagnostic tool for patients with chronic hepatitis C. However, the lack of reproducible results between laboratories and the relatively high proportion of false-positive results, has indicated the need for a standardized and reliable polymerase chain reaction assay. In the present study we have analyzed the performance of a commercial, HCV-RNA polymerase chain reaction assay based on a single, combined reverse transcription and amplification reaction and on the use of Uracil-N-glycosilase to prevent carry-over contamination (Amplicor HCV, Roche Molecular Systems). Methods: In this assay the amplification products are detected in microwell plates using biotinylated primer and the HRP avidin colorimetric system. Serum samples collected from 446 patients, including 181 with chronic active hepatitis C, 50 with autoimmune chronic hepatitis, 117 in hemodialysis, 30 asymptomatic carriers of anti-HCV and 68 with indeterminate serology (RIBA indeterminate results), as well as from 121 controls were tested with the commercial, single-step assay and with nested polymerase chain reaction. Both techniques use primers located within the 5' non-coding region of the HCV genome. Results: In all cases a good concordance was observed between the commercial, single-step assay and nested polymerase chain reaction which, for patients with chronic active hepatitis, showed a sensitivity and specificity of 100% and 99.3% for the former and of 98.8% and 100% for the latter, when compared to clinical diagnosis taken as the gold standard. Most of the 11 discordant samples were seen in the group of RIBA-indeterminate cases and in patients with chronic active hepatitis C. Further analysis of these cases, based on repeat testing and clinical data showed that 64% and 36% of the discrepancies were due, respectively, to nested polymerase chain reaction and Amplicor inconsistent reactions. In hemodialyzed patients, patients with autoimmune hepatitis and asymptomatic carriers of anti-HCV; both assays produced results which were consistent with the clinical diagnosis. In the former group, polymerase chain reaction was able to identify the presence of active viral replication in some antibody negative samples. Conclusions: Taken together, these results indicate that the commercial, single-step polymerase chain reaction assay has the same clinical sensitivity and specificity as nested polymerase chain reaction and that, because of its simplified procedures and fast turn-around time, it may be a valuable test for routine diagnostic applications.

KW - Chronic hepatitis

KW - Clinical samples

KW - HCV RNA

KW - PCR

UR - http://www.scopus.com/inward/record.url?scp=0030053266&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030053266&partnerID=8YFLogxK

U2 - 10.1016/S0168-8278(96)80183-8

DO - 10.1016/S0168-8278(96)80183-8

M3 - Article

VL - 24

SP - 33

EP - 37

JO - Journal of Hepatology

JF - Journal of Hepatology

SN - 0168-8278

IS - 1

ER -