Journal Information
Vol. 14. Issue 2.
Pages 147-152 (March - April 2010)
Share
Share
Download PDF
More article options
Vol. 14. Issue 2.
Pages 147-152 (March - April 2010)
Original article
Open Access
Signal to cut-off (S/CO) ratio and detection of HCV genotype 1 by real-time PCR one-step method: is there any direct relationship?
Visits
3321
Guilherme Albertoni
Corresponding author
albertonig@nefro.epm.br

Correspondence to: Av. Jandira, 1260, Indianópolis São Paulo – SP – Brazil CEP: 04080-006. Phone: 55 11 50556588/R. 39 Fax: 55 11 50556588.
, C.P. Arnoni, P.R.B. Araújo, F.O. Carvalho, J.A. Barreto
Colsan – Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil
This item has received

Under a Creative Commons license
Article information
Abstract
Bibliography
Download PDF
Statistics
Abstract
Background

Polymerase chain reaction (PCR) methods play an essential role in providing data related to diagnosis, monitoring and treatment of hepatitis C virus (HCV) infection. EIA results are reported as “reactive” or “non reactive” and EIA S/CO ratio may also be reported as “high” or “low.” This study aimed to evaluate the performance of a real-time RT-PCR and assess whether there is relationship between S/CO and PCR results.

Study Design and Methods

Sera from blood donors were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) and RT-PCR assay to detect HCV infection.

Results

The RT-PCR assay to genotypes 1a/b showed an acceptable linear response in serial dilutions. The samples were divided into two groups based on their serological results: group A – S/CO ratio < 3 (60 samples) and group B – S/CO ratio > 3 (41 samples). Viral loads were confirmed positive in group B samples in 90%, and in group A samples were confirmed positive in only 13% by RT-PCR.

Conclusion

The methodology used was able to detect the presence of RNA-HCV genotype I in 90% of the samples serologically positive in group B. All negative samples were sent to search for other genotypes of HCV (genotypes 2-6) and were confirmed as negative. These data suggests that these negative samples may have HCV RNA viral load below the detection limit of our test (310 IU/ mL), or a false positive result in serological test, or spontaneous viral clearance occurred.

Keywords:
HCV
HCV genotype 1
real-time PCR
ELISA
RNA extraction
Full text is only aviable in PDF
References
[1.]
M.J. Alter.
Transmission of HCV – Route, Dose and Titer.
New England Journal of Medicine, 173 (1994), pp. 784-786
[2.]
A.S.F. Lok, N.T. Gunaratnam.
Diagnosis of Hepatitis C.
Hepatology, 263 (1997), pp. 485-565
[3.]
L.P. Krug, V.R. Lunge, N. Ikuta.
Genótipos do vírus da hepatite C (VHC) no Rio Grande do Sul.
Laes & Haes, 19 (1997), pp. 78-82
[4.]
M.T.R. Justa, C. Terada.
Experiência do Laboratório Fleury na elucidação de perfis inconclusivos na sorologia para o vírus da hepatite C (HCV).
Congresso de Patologia e Medicina Laboratorial – São Paulo,
[5.]
S. Sreevatsan, J.B. Bookout, F.M. Ringpsis.
Algorithmic Approach to High-throughput Molecular Screening for Alpha interferon-resistant Genotypes in Hepatitis C Patients.
Journal of Clinical Microbiology, 36 (1998), pp. 1895-1901
[6]
World Health Organization. Epidemic and pandemic alert and response. Available at: http://www.who.int/csr/disease/hepatitis/whocdscssrlyo2003/en/index2.html. Accessed February 09, 2009.
[7.]
J.G. Mchutchinson, S.C. Gordon, E.R. Schiff.
Interferon Alpha-2b Alone or in Combination with Ribavirin as Initial Treatment for Chronic Hepatitis C. Hepatitis Interventional Therapy Group.
New England Journal of Medicine, 339 (1998), pp. 1485
[8.]
D.R. Gretch.
Use and Interpretation of HCV Diagnostic Tests in the Clinical Setting.
Clinics in Liver Disease, 1 (1997), pp. 553-557
[9.]
A.S. Muerhoff, L. Jiang, D.O. Shah, et al.
Detection of HCV core antigen in human serum and plasma with an automated chemiluminescent immunoassay.
Transfusion, 42 (2002), pp. 349-356
[10.]
M. Medina, E.R. Schiff, C. Hepatitis.
Diagnostic Assays.
Seminars in Liver Disease, 15 (1995), pp. 33-40
[11.]
J.Y.N. Lau, G.L. Davis, J. Kniffen, K. Qian.
Significance of Serum Hepatitis C virus RNA Levels in Chronic Hepatitis C.
Lancet, 341 (1993), pp. 1501-1504
[12]
Huber KR, Knapp M, Bauer K. Subtyping Hepatitis C Virus Clinical Chemistry 1995; 41:319-20.
[13.]
J.J. Lefrère, J. Coste, C. Defer.
Screening Blood Donstion for Viral Genomes: Multicenter Study of Real-Time Stimulation Using Pooled Samples on the Model of Hepatitis C Virus RNA Detection.
Transfusion, 38 (1998), pp. 915-923
[14.]
L.P. Krug, V.R. Lunge, N. Ikuta.
Hepatitis C Virus Genotypes in Southern Brazil.
Brazilian Journal of Medical and Biological Research, 29 (1996), pp. 1629-1632
[15.]
H. Ohto, S. Terezawa, N. Sasaki.
Transmission of Hepatitis C Virus from Mothers to Infants.
New England Journal of Medicine, 17 (1994), pp. 744-750
[16.]
S.L. Pogam, F. Dubois, R. Christen, C. Raby.
Comparison of DNA Enzyme Immunoassay and Line Probe Assays (InnoLipa HCV I and II) for hepatitis C virus genotyping.
Journal of Clinical Microbiology, 36 (1998), pp. 1461-1463
[17.]
T. Wang, S.S. Lee.
Hepatitis C: A review for primary care physicals.
CMAJ, 174 (2006), pp. 649-659
[18.]
S.M. Kamal.
Acute hepatitis C: a systematic review.
Am. J. Gastrol, 103 (2008), pp. 1283-1297
[19.]
C.S. Chevaliez, J.M. Pawlotsky.
Practical use of hepatitis C virus kinetics monitoring in the treatment of chronic hepatitis.
Journal of Viral Hepatitis, 14 (2007), pp. 77-81
[20.]
C.J. Fregeau, C.M. Lett, J. Elliott, C. Yensen, R.M. Fourney.
Automated processing of forensic casework samples using robotic workstations equipped with nondisposable tips: contamination prevention.
J. Forensic. Sci, 53 (2008), pp. 632-651
[21.]
J.L. Gerberding, D.W. Fleming, D.E. Snider.
Guidelines for laboratory testing and result reporting of antibody to hepatitis C virus. Epidemiology Program Office, Centers for Disease control and Prevention (CDC).
Department of Health and Human Services, 52 (2003), pp. 1-15
[22.]
S.A. Whalley, J.M. Murray, D. Brown, et al.
Kinetics of acute hepatitis B virus infection in humans.
J. Exp. Med, 193 (2001), pp. 847-854
[23.]
A. Humar, D. Kumar, B. Boivin, A.M. Caliendo.
Cytomegalovirus (CMV) virus load kinetics to predict recurrent disease in solid-organ transplant patients with CMV disease.
J. Infect. Dis, 186 (2002), pp. 829-833
[24.]
P.J. Snijders, E.A.J. Van Den Brul, C.J. Meijer.
The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity.
J. Pathol, 201 (2003), pp. 1-6
[25.]
K. Biedermann, N. Dandachi, M. Trattner, et al.
Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue.
J. Clin. Microbiol, 42 (2004), pp. 3758-3765
[26.]
H.J. Wagner, Y.C. Cheng, M.H. Huls, et al.
Prompt versus preemptive intervention for EBV lymphoproliferative disease.
Blood, 103 (2004), pp. 3979-3981
[27.]
F. Watzinger, M. Suda, S. Preuner, et al.
Real –time quantitative PCR assay for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.
J. Clin. Microbiol, 42 (2004), pp. 5189-5198
[28.]
P.R. Selvin.
Fluorescence resonance energy transfer.
Methods Enzymol, 246 (1995), pp. 300-334
[29.]
P.M. Holland, R.D. Abramson, R. Watson, D.H. Gelfand.
Detection of specific polymerase chain reaction product by utilizing the 50-30 exonuclease activity of thermos aquaticus DNA polymerase.
Proc. Natl. Acad. Sci. USA, 88 (1991), pp. 7276-7280
[30.]
M. Beld, R. Sentjens, S. Rebers.
Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantification of hepatitis C virus RNA in plasma and serum: Conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, version 2.0, assay.
J. Clin. Microbiol, 40 (2002), pp. 788-793
[31.]
S.C. Lee, A. Antony, N. Lee, et al.
Improved version 2.0 qualitative and quantitative AMPLICOR reverse transcription- PCR tests for hepatitis C virus RNA: Calibration to international units, enhance genotype reactivity, and performance characteristics.
J. Clin. Microbiol, 38 (2000), pp. 4171-4179
[32.]
S. Kawai, O. Yokosuba, T.J. Kanda.
Quantification of hepatitis C virus by TaqMan PCR: comparison with HCV Amplicor Monitor assay.
J. Med. Virol, 58 (1999), pp. 121-126
[33.]
F. Komurian-Pradel, G. Paranhos-Baccala, M. Sosoyer.
Quantification of HCV RNA using real-time PCR and fluorimetry.
J. Virol. Methods, 95 (2001), pp. 111-119
[34.]
M. Puig, K. Mihalik, M.Y. Yu, S.M. Feinstone, M.E. Major.
Sensitive and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-tiem PCR assays.
J. Virol. Methods, 105 (2002), pp. 253-263
[35.]
T. Takeuchi, A. Katsume, T. Tanaka, et al.
Real-time detection system for quantification of hepatitis C virus genome.
Gastroenterology, 116 (1999), pp. 636-642
[36.]
L.D. Trani, B. Bedini, I. Donatelli, et al.
A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control.
BMC. Infectious Diseases, 87 (2006), pp. 1-8
[37.]
B.C. Arruda, R.A. Lira, P. Loureiro, et al.
Evaluation of real time PCR technique to diagnosis of human T-lymphotropic virus type I (HTLV-I) in patients in hematology at Fundação Hemope Hospital, in northeastern Brazil.
Rev. Bras. Hematol. Hemoter, 30 (2008), pp. 384-389
[38.]
L.N. Wang, J.M. Wu, W. Deng, et al.
Lyophilized standards for the calibration of real time PCR assay for hepatitis C virus RNA.
J. Chin. Med., 119 (2006), pp. 1910-1914
[39.]
M.F. Meyer, M. Lehmann, M. Cornberg, et al.
Clearance of low levels of HCV viremia in the absence of a strong adaptative immune response.
Virology Journal, 11 (2007), pp. 1-11
[40.]
L.A. Kondili, A. Constantino, U. Villano, et al.
Infection rate and spontaneous seroreversion of anti-hepatitis C virus during the natural course of hepatitis C virus infection in the general population.
Liver Disease, 50 (2002), pp. 693-696
[41]
Gonçalves FL, Gonçalves NSL, Cavalheiro NP. I Consenso da Sociedade Paulista de Infectologia para manuseio e terapia da Hepatite C. Available at: http://www.praticahospitalar.com.br/pratica%2027/Concenso/3.html, 2002. Accessed February 17, 2009.
Copyright © 2010. Elsevier Editora Ltda.. All rights reserved
Download PDF
The Brazilian Journal of Infectious Diseases
Article options
Tools