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
3291
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
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