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Farag MMS, Sofy AR, Mousa AA, Ahmed MA, Alganzory MR. Molecular Assay and Genotyping of Hepatitis C Virus among Infected Egyptian and Saudi Arabian Patients. Virology (Auckl) 2015; 6:1-10. [PMID: 26512201 PMCID: PMC4603572 DOI: 10.4137/vrt.s32016] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Revised: 09/03/2015] [Accepted: 09/10/2015] [Indexed: 01/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection is a major health problem recognized globally. HCV is a common cause of liver fibrosis that may lead to liver cirrhosis or hepatocellular carcinoma. The aim of this study was to estimate the prevalence of HCV infection and genotyping among Egyptian and Saudi Arabian chronic patients using different molecular techniques. HCV RNA viral load was assessed by real-time polymerase chain reaction (RT-PCR) technology. For HCV genotyping, RT-PCR hybridization fluorescence-based method and reverse hybridization line probe assay (INNO-LiPA) were used. A total of 40 anti-HCV-positive patients with chronic hepatitis C were examined for HCV RNA, genotyping, and different laboratory investigations. In the present study, HCV genotypes 4, mixed 4.1b, and 1 were detected in patients of both countries, while genotype 2 was only detected in Saudi Arabian patients. Genotyping methods for HCV showed no difference in the classification at the genotype level. With regard to HCV subtypes, INNO-LiPA assay was a reliable test in HCV genotyping for the detection of major genotypes and subtypes, while RT-PCR-based assay was a good test at the genotype level only. HCV genotype 4 was found to be the predominant genotype among Egyptian and Saudi Arabian chronic patients. In conclusion, data analysis for detecting and genotyping HCV was an important factor for understanding the epidemiology and treatment strategies of HCV among Egyptian and Saudi Arabian chronic patients.
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Affiliation(s)
- Mohamed MS Farag
- Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
| | - Ahmed R Sofy
- Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
| | - Adel A Mousa
- Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt
| | - Mohamed A Ahmed
- Clinical Pathology Department, Military Medical Academy, Cairo, Egypt
| | - Mohamed R Alganzory
- Basic Science Department, College of Dentistry, Majma’ah University, Al Majma’ah, Kingdom of Saudi Arabia
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Bagaglio S, Uberti-Foppa C, Di Serio C, Trentini F, Andolina A, Hasson H, Messina E, Merli M, Porrino L, Lazzarin A, Morsica G. Dynamic of Mixed HCV Infection in Plasma and PBMC of HIV/HCV Patients Under Treatment With Peg-IFN/Ribavirin. Medicine (Baltimore) 2015; 94:e1876. [PMID: 26512601 PMCID: PMC4985415 DOI: 10.1097/md.0000000000001876] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2015] [Revised: 09/25/2015] [Accepted: 09/29/2015] [Indexed: 02/07/2023] Open
Abstract
The extent of mixed hepatitis C virus (HCV) genotype in different compartments (plasma and peripheral blood mononuclear cell, PBMC) and possible association with treatment efficacy in HIV/HCV coinfected patients remains to be unknown.The objective of this study was to elucidate the frequency of mixed genotype infection (MG), its profile in different compartments during anti-HCV treatment, and the possible influence of different genotypes on the response rate.The compartmentalization of HCV population was investigated by next-generation sequencing in 19 HIV/HCV coinfected patients under anti-HCV treatment with peginterferon/ribavirin (P-R). Ten individuals were nonresponder (NR) or relapser (RE) to P-R treatment and 9 had a sustained virological response (SVR).Eleven/nineteen (58%) patients had MG in plasma compartment. Ten or 12 patients infected by a difficult to treat genotype (DTG) 1 or 4 as dominant strain, had an MG, whereas only 1/7 individuals infected by easy to treat genotype (ETG) harbored a mixed genotype, P = 0.006. HCV-RNA was more frequently detected in PBMC of NR (10/10) than in those of SVR (5/9), P = 0.032. Mixed genotype infection was detected in 6/15 (40%) PBMC-positive cases and was not associated with P-R treatment response. By multivariate analysis, MG in plasma samples was the most important viral factor affecting the treatment response (P = 0.0237).Detection of MG in plasma of HIV/HCV coinfected patients seems to represent the major determinant of response to P-R treatment. This finding may have important clinical implication in light of the new therapeutic approach in HIV/HCV coinfected individuals suggesting that combination treatment with direct acting antivirals could be less effective in MG.
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Affiliation(s)
- Sabrina Bagaglio
- Infectious Diseases Department, Scientific Institute Ospedale San Raffaele, Via Stamira d'Ancona 20, 20127 Milan, Italy (SB, CU-F, AA, HH, EM, MM, AL, GM); Vita-Salute University Via Olgettina 58, 20132 Milan, Italy (SB, AL); University Centre for Statistics in the Biomedical Sciences (CUSSB) Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy (CDS, FT); L. Bocconi University, Via R. Sarfatti 25, 20136 Milan, Italy (FT); Mol Med Spa, Via Olgettina 58, 20132 Milan, Italy (LP)
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Athar MA, Xu Y, Xie X, Xu Z, Ahmad V, Hayder Z, Hussain SS, Liao Y, Li Q. Rapid detection of HCV genotyping 1a, 1b, 2a, 3a, 3b and 6a in a single reaction using two-melting temperature codes by a real-time PCR-based assay. J Virol Methods 2015; 222:85-90. [PMID: 26068393 DOI: 10.1016/j.jviromet.2015.05.013] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2015] [Revised: 05/26/2015] [Accepted: 05/26/2015] [Indexed: 12/18/2022]
Abstract
The genotype of the hepatitis C virus (HCV) is an important indicator for antiviral therapeutic response. We hereby described development of a rapid HCV genotyping approach that enabled the identification of the six most common HCV subtypes of Asia, i.e., 1a, 1b, 2a, 3a, 3b, and 6a, in a single reaction. Using two dual-labeled, self-quenched probes that target the core region of the HCV genome, the exact subtype could be accurately identified by two-melting temperature codes determined from the two respective probes in a real-time PCR assay. Analytical sensitivity studies using armored RNA samples representing each of the six HCV subtypes showed that 5 copies/reaction of HCV RNA could be detected. The assay was evaluated using 244 HCV-positive serum samples and the results were compared with sequencing analysis. Of the 224 samples, subtype 3a (127, 52.3%) was the dominant, followed by 1b (51, 20.9%), 3b (47, 19.3%), 2a (8, 3.3%), 6a (4, 1.6%) and the least was subtype 1a (1, 0.4%). Moreover, 6 (2.5%) mixed infection samples were also detected. These results were fully concordant with sequencing analysis. We concluded that this real-time PCR-based assay could provide a rapid and reliable tool for routine HCV genotyping in most Asian countries.
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Affiliation(s)
- Muhammad Ammar Athar
- The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China
| | - Ye Xu
- The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China
| | - Xiaoting Xie
- The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China
| | - Zhenxing Xu
- Xiamen Hospital of Traditional Chinese Medicine, Xiamen 361005, Fujian, China
| | - Vakil Ahmad
- Division of Health Biotechnology, National Institute for Biotechnology & Genetic Engineering, P.O. Box 577, Faisalabad, Punjab, Pakistan
| | - Zulfiqar Hayder
- Department of Pathology, Quid-e-Azam Medical College, Bahawalpur, Punjab, Pakistan
| | - Syed Sajid Hussain
- Department of Pathology, Quid-e-Azam Medical College, Bahawalpur, Punjab, Pakistan
| | - Yiqun Liao
- State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361005, Fujian, China.
| | - Qingge Li
- The State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics, Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong 518057, China.
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McCormick AL, Macartney MJ, Abdi-Abshir I, Labbett W, Smith C, Irish D, Webster DP, Haque T. Evaluation of sequencing of HCV core/E1, NS5A and NS5B as a genotype predictive tool in comparison with commercial assays targeting 5'UTR. J Clin Virol 2015; 66:56-9. [PMID: 25866338 DOI: 10.1016/j.jcv.2015.03.006] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2014] [Revised: 02/24/2015] [Accepted: 03/06/2015] [Indexed: 02/05/2023]
Abstract
BACKGROUND Hepatitis C virus (HCV) genotyping is required for tailoring the dose and duration of antiviral therapy, predicting virological response rates, and selecting future treatment options. OBJECTIVE To establish whether baseline genotypes, performed by INNO-LiPA Version 1.0 (v1.0), before 2008, were valid for making treatment decisions now or whether genotypic determination should be repeated. Furthermore, to evaluate concordance between Abbott RealTime genotype II assay (RT) and genotyping by sequencing HCV C/E1, NS5A, NS5B. STUDY DESIGN Genotyping by RT and sequencing was performed on paired historic and current specimens from 50 patients previously baseline genotyped using INNO-LiPA. RESULTS Of 100 samples from 50 patients, ≥ 2 of HCV genomic target regions yielded a sequence that was suitable for genotyping, with 100% concordance, providing no evidence of recombination events. Genotype and subtype prediction based on RT and sequencing agreed in 62.8% historic and 72.7% current specimens, with a kappa coefficient score of 0.48 and 0.76, respectively. LiPA could not subtype 46% of HCV gt1 infections, and LiPA subgenotype was only in agreement with RT and sequencing in 28.6% cases, where matched baseline and historic specimens were available. Three patients were indeterminate by RT, and five patients with HCV gt1 infections could not be subtyped by RT. However, RT revealed mixed infections in five patients where sequencing detected only single HCV infection at 20% threshold. CONCLUSION Genotyping by sequencing, exhibited excellent concordance, with moderate to good agreement with RT, and could resolve RT indeterminates and subtype HCV-gt1 infections not possible by LiPA.
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Affiliation(s)
- Adele L McCormick
- Department of Virology, Royal Free London NHS Foundation Trust, London, UK.
| | | | - Ikran Abdi-Abshir
- Department of Virology, Royal Free London NHS Foundation Trust, London, UK
| | - Wendy Labbett
- Department of Virology, Royal Free London NHS Foundation Trust, London, UK
| | - Colette Smith
- Department of Infection and Population Health, University College London, UK
| | - Dianne Irish
- Department of Virology, Royal Free London NHS Foundation Trust, London, UK
| | - Daniel P Webster
- Department of Virology, Royal Free London NHS Foundation Trust, London, UK
| | - Tanzina Haque
- Department of Virology, Royal Free London NHS Foundation Trust, London, UK
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Optimization and application of subtype specific polymerase chain reaction for detection and identification of mixed subtypes of hepatitis C virus in patients with renal disease. J Virol Methods 2012; 186:113-8. [DOI: 10.1016/j.jviromet.2012.06.023] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2011] [Revised: 06/26/2012] [Accepted: 06/27/2012] [Indexed: 02/04/2023]
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Uccellini L, Tseng FC, Monaco A, Shebl FM, Pfeiffer R, Dotrang M, Buckett D, Busch MP, Wang E, Edlin BR, Marincola F, O’Brien TR. HCV RNA levels in a multiethnic cohort of injection drug users: human genetic, viral and demographic associations. Hepatology 2012; 56:86-94. [PMID: 22331649 PMCID: PMC3369001 DOI: 10.1002/hep.25652] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/07/2011] [Accepted: 01/28/2012] [Indexed: 12/28/2022]
Abstract
UNLABELLED In patients with chronic hepatitis C, the hepatitis C virus (HCV) RNA level is an important predictor of treatment response. To explore the relationship of HCV RNA with viral and demographic factors, as well as IL28B genotype, we examined viral levels in an ethnically diverse group of injection drug users (IDUs). Between 1998 and 2000, the Urban Health Study (UHS) recruited IDUs from street settings in San Francisco Bay area neighborhoods. Participants who were positive by HCV enzyme immunoassay were tested for HCV viremia by a branched-chain DNA assay. HCV genotype was determined by sequencing the HCV nonstructural 5B protein region. For a subset of participants, IL28B rs12979860 genotype was determined by Taqman. Among 1,701 participants with HCV viremia, median age was 46 years and median duration of injection drug use was 26 years; 56.0% were African American and 34.0% were of European ancestry (non-Hispanic). Human immunodeficiency virus type 1 (HIV-1) prevalence was 13.9%. The overall median HCV RNA level was 6.45 log(10) copies/mL. In unadjusted analyses, higher levels were found with older age, male gender, African-American ancestry, hepatitis B virus infection, HIV-1 infection, and IL28B rs12979860-CC genotype; compared to participants infected with HCV genotype 1, HCV RNA was lower in participants with genotypes 3 or 4. In an adjusted analysis, age, gender, racial ancestry, HIV-1 infection, HCV genotype, and IL28B rs12979860 genotype were all independently associated with HCV RNA. CONCLUSION The level of HCV viremia is influenced by a large number of demographic, viral, and human genetic factors.
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Affiliation(s)
- Lorenzo Uccellini
- Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center and Trans-NIH Center for Human Immunology, NIH, Bethesda, Maryland
- Institute of Infectious and Tropical Diseases, University of Milan, L. Sacco Hospital, Milan, Italy
| | - Fan-Chen Tseng
- Division of Cancer Epidemiology and Genetics, National Cancer Institute
| | - Alessandro Monaco
- Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center and Trans-NIH Center for Human Immunology, NIH, Bethesda, Maryland
| | - Fatma M. Shebl
- Division of Cancer Epidemiology and Genetics, National Cancer Institute
| | - Ruth Pfeiffer
- Division of Cancer Epidemiology and Genetics, National Cancer Institute
| | | | - Dianna Buckett
- Division of Cancer Epidemiology and Genetics, National Cancer Institute
| | - Michael P. Busch
- University of California, San Francisco, CA
- Blood Systems Research Institute, San Francisco, CA
| | - Ena Wang
- Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center and Trans-NIH Center for Human Immunology, NIH, Bethesda, Maryland
| | - Brian R. Edlin
- University of California, San Francisco, CA
- SUNY Downstate College of Medicine, Brooklyn, NY and Center for the Study of Hepatitis C, Weill Medical College of Cornell University, New York, NY
| | - Francesco Marincola
- Infectious Disease and Immunogenetics Section, Department of Transfusion Medicine, Clinical Center and Trans-NIH Center for Human Immunology, NIH, Bethesda, Maryland
| | - Thomas R. O’Brien
- Division of Cancer Epidemiology and Genetics, National Cancer Institute
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Butt S, Idrees M, Ur Rehman I, Akbar H, Shahid M, Afzal S, Younas S, Amin I. Mixed genotype infections with hepatitis C virus, Pakistan. Emerg Infect Dis 2011; 17:1565-7. [PMID: 21801655 PMCID: PMC3381542 DOI: 10.3201/eid1708.100950] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
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Idrees M, Ur Rehman I, Manzoor S, Akbar H, Butt S, Afzal S, Yousaf MZ, Hussain A. Evaluation of three different hepatitis C virus typing methods for detection of mixed-genotype infections. J Dig Dis 2011; 12:199-203. [PMID: 21615874 DOI: 10.1111/j.1751-2980.2011.00496.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVE To evaluate the clinical applicability of an eligible assay for the true prevalence of hepatitis C virus (HCV) mixed-genotype infections. METHODS A newly developed HCV genotyping method targeting all six major HCV genotypes and 12 subtypes, restriction fragment length polymorphism (RFLP) and a serotyping assay were utilized for the detection of HCV mixed-genotype infections using known HCV genotypes and unknown samples. RESULTS In a defined mix of HCV genotypes, a genotype present at levels as low as 8.3% was detected by our newly developed assay, showing a threefold increase in sensitivity over that of direct deoxyribonucleic (DNA) sequencing. A comparative study of the accuracy among the three genotyping methods was carried out on samples obtained from 50 thalassemic patients who received multiple blood transfusions. The results showed that viruses in approximately 42% of the samples from this group were determined to be infected with mixed genotypes by our newly developed method. A serotyping assay and RFLP analysis, performed with poor results, could identify only 18% and 10% of mixed-genotype infections, respectively. CONCLUSION The newly developed assay may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections due to its higher level of sensitivity.
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Affiliation(s)
- Muhammad Idrees
- Division of Molecular Virology and Molecular Diagnostics, National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
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Diversity of hepatitis C virus genotypes among intravenous heroin users in Taiwan. Am J Med Sci 2011; 341:110-2. [PMID: 20924285 DOI: 10.1097/maj.0b013e3181f5582f] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
INTRODUCTION Limited information is available about genotypes of hepatitis C virus (HCV) in intravenous heroin users in Taiwan. The purpose of this study was to examine the concordance of the detection of antibody to HCV and HCV-RNA and to determine the distribution of HCV genotypes in male intravenous heroin users. METHODS This was a cross-sectional study. The study population included 274 intravenous heroin drug users newly sentenced in a male prison in central Taiwan from November 2004 to February 2005, whose antibodies to HCV were positive, and antibodies to human immunodeficiency virus were negative. The mean age was 33.9 years (standard deviation, 7.8). The molecular diagnosis used to identify HCV-RNA was PCR. RESULTS Among 274 subjects, 214 subjects were found to contain HCV-RNA. Positive predictive value of HCV infection using antibody to HCV as an indicator was 78.1%. Among 214 subjects, HCV genotype 2a was the most predominant (58.9%, n = 126), followed by 1a (17.3%, n = 37), 1b (14.5%, n = 31), 2b (8.9%, n = 19) and 1a + 2b (0.4%, n = 1). Age-specific analysis also showed genotype 2a was the most prominent genotype among the 4 age groups, with the highest prevalence in groups aged 20 to 29 years and 30 to 39 years (53.3% and 67.6%, respectively). CONCLUSIONS The concordance of antibody to HCV and HCV-RNA is remarkable in selected high-risk groups. HCV genotype 2a is the most prevalent in male intravenous heroin users in central Taiwan, especially in aged 20 to 29 years and aged 30 to 39 years.
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Koletzki D, Dumont S, Vermeiren H, Fevery B, De Smet P, Stuyver LJ. Development and evaluation of an automated hepatitis C virus NS5B sequence-based subtyping assay. Clin Chem Lab Med 2010; 48:1095-102. [DOI: 10.1515/cclm.2010.236] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
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Abstract
International standardization and coordination of the nomenclature of variants of hepatitis C virus (HCV) is increasingly needed as more is discovered about the scale of HCV-related liver disease and important biological and antigenic differences that exist between variants. Consistency in numbering is also increasingly required for functional and clinical studies of HCV. For example, an unambiguous method for referring to amino acid substitutions at specific positions in NS3 and NS5B coding sequences associated with resistance to specific HCV inhibitors is essential in the investigation of antiviral treatment. Inconsistent and inaccurate numbering of locations in DNA and protein sequences is becoming a problem in the HCV scientific literature.A group of experts in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and the Los Alamos National Laboratory (United States), convened to reexamine the status of HCV genotype nomenclature, resolve conflicting genotype or subtype names among described variants of HCV, and draw up revised criteria for the assignment of new genotypes as they are discovered in the future. They also discussed how HCV sequence databases could introduce and facilitate a standardized numbering system for HCV nucleotides, proteins, and epitopes.A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name reassignments to create consistency in nomenclature. A consensus proposal was drawn up for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. The proposed numbering system was adapted from the Los Alamos HIV database, with elements from the hepatitis B virus numbering system. The system comprises both nucleotides and amino acid sequences and epitopes, and uses the full-length genome sequence of isolate H77 (accession number AF009606) as a reference. It includes a method for numbering insertions and deletions relative to this reference sequence.
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Affiliation(s)
- Carla Kuiken
- Los Alamos National Laboratory, Los Alamos, NM, USA
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Scott JD, Garland N. Chronic liver disease in Aboriginal North Americans. World J Gastroenterol 2008; 14:4607-15. [PMID: 18698674 PMCID: PMC2738784 DOI: 10.3748/wjg.14.4607] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/06/2008] [Revised: 05/28/2008] [Accepted: 06/04/2008] [Indexed: 02/06/2023] Open
Abstract
A structured literature review was performed to detail the frequency and etiology of chronic liver disease (CLD) in Aboriginal North Americans. CLD affects Aboriginal North Americans disproportionately and is now one of the most common causes of death. Alcoholic liver disease is the leading etiology of CLD, but viral hepatitis, particularly hepatitis C, is an important and growing cause of CLD. High rates of autoimmune hepatitis and primary biliary cirrhosis (PBC) are reported in regions of coastal British Columbia and southeastern Alaska. Non-alcoholic liver disease is a common, but understudied, cause of CLD. Future research should monitor the incidence and etiology of CLD and should be geographically inclusive. In addition, more research is needed on the treatment of hepatitis C virus (HCV) infection and non-alcoholic fatty liver disease (NAFLD) in this population.
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Investigation of putative multisubtype hepatitis C virus infections in vivo by heteroduplex mobility analysis of core/envelope subgenomes. J Virol 2008; 82:7524-32. [PMID: 18495766 DOI: 10.1128/jvi.02220-07] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The frequency that multiple different subtypes of hepatitis C virus (HCV) simultaneously infect a given individual is controversial. To address this question, heteroduplex mobility analysis (HMA) of portions of the HCV core and envelope 1 region was optimized for sensitive and specific detection of mixtures of HCV genomes of different genotype or subtype. Using the standard HCV genotyping approach of 5'-untranslated region (UTR) analysis, 28 of 374 (7.5%) chronic hepatitis C research subjects were classified as having either multiple-subtype HCV infections (n = 21) or switching HCV subtypes over time (n = 7), the latter pattern implying viral superinfection. Upon retesting of specimens by HMA, 25 of 28 multiple-subtype results could not be reproduced. All three patients with positive results were injection drug users with potential multiple HCV exposures. To address the hypothesis of tissue sequestration of multiple-subtype HCV infections, liver (n = 22), peripheral blood mononuclear cell (n = 13), perihepatic lymph node (n = 16), and serum (n = 19) specimens from 23 subjects with end-stage hepatitis C were collected and analyzed by the HMA technique. Whereas 5'-UTR results implicated mixed-subtype HCV infections in 2 subjects, HMA testing revealed no evidence of a second HCV subtype in any tissue compartment (0 of 70 compartments [0%]) or within any given subject (0 of 23 subjects [0%]). In summary, a large proportion of mixed-genotype and switching-genotype patterns generated by 5'-UTR analysis were not reproducible using the HMA approach, emphasizing the need for additional study.
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Mao H, Lu Z, Zhang H, Liu K, Zhao J, Jin G, Gu S, Yang M. Colorimetric oligonucleotide array for genotyping of hepatitis C virus based on the 5′ non-coding region. Clin Chim Acta 2008; 388:22-7. [DOI: 10.1016/j.cca.2007.09.009] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2007] [Revised: 09/12/2007] [Accepted: 09/13/2007] [Indexed: 10/22/2022]
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15
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Hejazi MS, Ghotaslou R, Hagh MF, Sadigh YM. Genotyping of Hepatitis C Virus in Northwest of Iran. ACTA ACUST UNITED AC 2007. [DOI: 10.3923/biotech.2007.302.308] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
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16
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Bouchardeau F, Cantaloube JF, Chevaliez S, Portal C, Razer A, Lefrère JJ, Pawlotsky JM, De Micco P, Laperche S. Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay. J Clin Microbiol 2007; 45:1140-5. [PMID: 17251399 PMCID: PMC1865817 DOI: 10.1128/jcm.01982-06] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2006] [Revised: 11/12/2006] [Accepted: 01/13/2007] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) isolates have been classified into six main genotypes. Genotyping methods, and especially the widely used line probe assay (LiPA), are frequently based on the 5'-untranslated region (5'UTR). However, this region is not appropriate for discriminating HCV strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1. We investigated the capacity of a novel LiPA (Versant HCV Genotype 2.0 assay) based on the simultaneous detection of 5'UTR and Core regions for genotypes 1 and 6 to provide correct HCV genotypes (characterized with a phylogenetic analysis) in a set of HCV strains mainly encountered in Western countries. The improvement was assessed by comparing the results to those obtained with the previous version of the assay. Of the 135 tested samples, 64.7% were concordant for genotype group and subtype with sequencing reference results using the Versant HCV Genotype 2.0 assay versus 37.5% with the previous version. The yield was mainly related to a better characterization of genotype 1, since the accuracy, tested in 62 genotype 1 samples, increased from 45.2% with the first version to 96.8% with the new one. However, this new version necessitates a specific PCR and could no longer be used after 5'UTR PCR used for current HCV infection diagnosis. Moreover, the information provided by 5'UTR hybridization is not reliable for correctly identifying the diversity within genotypes 2 and 4. Thus, the Versant HCV Genotype 2.0 assay remains a useful tool for clinical practice when only the discrimination between major HCV genotypes is necessary.
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Affiliation(s)
- Françoise Bouchardeau
- Institut National de la Transfusion Sanguine, 6 Rue Alexandre-Cabanel, 75015 Paris, France
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Abstract
Hepatitis C virus is an RNA virus that is associated with chronic infection in the majority of people infected. Chronic infection with hepatitis C virus is the cause of significant morbidity and mortality worldwide and is associated with a large spectrum of liver disease including cirrhosis and hepatocellular carcinoma. End-stage liver disease due to chronic hepatitis C virus infection is currently the leading indication for liver transplantation in the USA. Hepatitis C virus genotyping of viral isolates circulating in the blood during chronic infection has become an important part of hepatitis C virus monitoring in chronically infected patients, and is useful as a prognostic indicator and to direct duration of therapy. This review will summarize information on hepatitis C genotyping, describe the limitations of current commercially available methods, give information on more recently developed methods, and provide a look to the future in terms of where advances in hepatitis C virus genotyping assays need to be made.
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Affiliation(s)
- Karen Weck
- University of North Carolina, Campus Box 7525, Chapel Hill, NC 27599-7525, USA.
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Lee CM, Lu SN, Hung CH, Tung WC, Wang JH, Tung HD, Chen CH, Hu TH, Changchien CS, Chen WJ. Hepatitis C virus genotypes in southern Taiwan: prevalence and clinical implications. Trans R Soc Trop Med Hyg 2006; 100:767-74. [PMID: 16443243 DOI: 10.1016/j.trstmh.2005.10.008] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2005] [Revised: 10/20/2005] [Accepted: 10/21/2005] [Indexed: 12/20/2022] Open
Abstract
The role of hepatitis C virus (HCV) genotypes in the development of hepatocellular carcinoma (HCC) is still controversial. To determine the distribution and clinical implications of HCV genotypes in southern Taiwan, we analysed 418 patients with chronic HCV infections. HCV genotypes were determined using an HCV Line Probe Assay. The predominant HCV genotype was 1b (45.5%), followed by 2a/2c (30.9%) and 2b (6.9%). The prevalence of genotype 1b in HCC patients (60.3%) was significantly higher than in those with liver cirrhosis (38.7%) and chronic hepatitis (38.7%) (P=0.003 and P<0.001, respectively). Patients with chronic HCV 2a/2c infection had higher alanine aminotransferase (ALT) levels than those with chronic HCV 1b infection (P<0.001). Univariate analysis revealed that disease severity was significantly correlated with older age, genotype 1b, lower ALT levels and lower viral load. Based on multiple logistic regression analysis, after adjusting for age and serum HCV RNA levels, HCV 1b infection was still a significant risk factor for HCC. In conclusion, the predominant genotypes in southern Taiwan were 1b and 2a/2c, and disease severity was associated with genotype 1b.
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Affiliation(s)
- Chuan-Mo Lee
- Division of Hepatogastroenterology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, 123 Ta Pei Road, Kaohsiung, Taiwan.
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Lindh M, Hannoun C. Genotyping of hepatitis C virus by Taqman real-time PCR. J Clin Virol 2006; 34:108-14. [PMID: 16157261 DOI: 10.1016/j.jcv.2005.02.002] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2004] [Revised: 01/06/2005] [Accepted: 02/03/2005] [Indexed: 11/21/2022]
Abstract
BACKGROUND Genotype of hepatitis C virus (HCV) is of major importance for the outcome of treatment. The response rate is considerably lower for genotype 1, the predominant genotype in western countries. OBJECTIVES To develop and evaluate a new, simple method for genotyping of HCV based on real-time polymerase chain reaction (PCR) and Taqman probes targeting the 5' non-coding region. STUDY DESIGN The method was compared with Innolipa on 220 serum samples representing genotypes 1-4, and was applied on a further 614 clinical samples. RESULTS Taqman typing of the 220 samples showed genotype 1 in 69, genotype 2 in 58, genotype 3 in 57 and genotype 4 in 19, while 17 were non-reactive. There was a complete concordance with Innolipa with the exception of seven samples, which were of genotype 1 by Taqman, but genotype 4 by Innolipa. Sequencing of these samples showed a subtype 4 variant which differed at two positions compared with subtypes 4b/c/d, which are targeted by the probe. By adding a modified probe, these genotype 4 variants could also be identified. Out of 614 consecutive clinical samples, 524 could be typed by the Taqman assay; 45.2% were genotype 1, 19.3% genotype 2, 33.8% genotype 3 and 1.7%, genotype 4. CONCLUSION The method was overall accurate and provides an attractive alternative for genotyping because processing time and costs are significantly reduced. Inclusion of probes targeting genotypes 5 and 6 is required for the method to be useful in areas where these genotypes are present.
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Affiliation(s)
- Magnus Lindh
- Department of Clinical Virology, Guldhedsgatan 10 B, 413 46 Göteborg, Sweden.
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Simmonds P, Bukh J, Combet C, Deléage G, Enomoto N, Feinstone S, Halfon P, Inchauspé G, Kuiken C, Maertens G, Mizokami M, Murphy DG, Okamoto H, Pawlotsky JM, Penin F, Sablon E, Shin-I T, Stuyver LJ, Thiel HJ, Viazov S, Weiner AJ, Widell A. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005; 42:962-73. [PMID: 16149085 DOI: 10.1002/hep.20819] [Citation(s) in RCA: 1081] [Impact Index Per Article: 54.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
International standardization and coordination of the nomenclature of variants of hepatitis C virus (HCV) is increasingly needed as more is discovered about the scale of HCV-related liver disease and important biological and antigenic differences that exist between variants. A group of scientists expert in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and Los Alamos (United States), met to re-examine the status of HCV genotype nomenclature, resolve conflicting genotype or subtype names among described variants of HCV, and draw up revised criteria for the assignment of new genotypes as they are discovered in the future. A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name re-assignments to create consistency in nomenclature. The paper also contains consensus proposals for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. A proposal was made that HCV variants be classified into 6 genotypes (representing the 6 genetic groups defined by phylogenetic analysis). Subtype name assignment will be either confirmed or provisional, depending on the availability of complete or partial nucleotide sequence data, or remain unassigned where fewer than 3 examples of a new subtype have been described. In conclusion, these proposals provide the framework by which the HCV databases store and provide access to data on HCV, which will internationally coordinate the assignment of new genotypes and subtypes in the future.
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Affiliation(s)
- Peter Simmonds
- Centre for Infectious Diseases, University of Edinburgh, Summerhall, Edinburgh, United Kingdom.
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Domiati-Saad R, Scheuermann RH. Nucleic acid testing for viral burden and viral genotyping. Clin Chim Acta 2005; 363:197-205. [PMID: 16098959 DOI: 10.1016/j.cccn.2005.05.049] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2005] [Revised: 05/09/2005] [Accepted: 05/16/2005] [Indexed: 11/23/2022]
Abstract
BACKGROUND Molecular diagnostics plays an important role in the diagnosis and clinical management of a wide array of infectious diseases. METHODS Advances in molecular technology and methods of detecting nucleic acid sequences have revolutionized the field of virology. These developments are reflected by the rapid diagnosis and monitoring of viral agents as well as assessment of clinical disease associated with viral infections. In addition to many commercially available molecular based assays, many laboratories offer in-house developed assays for a variety of viral targets. The introduction of real-time PCR technology has made a large impact on virology testing. CONCLUSIONS The role of real-time PCR for the diagnosis of viral infections is enhanced by the accuracy, rapidity and ability to quantitate viral target sequences.
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Affiliation(s)
- Rana Domiati-Saad
- Department of Pathology, Baylor University Medical Center, Dallas, TX 75246, USA.
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Kim YJ, Kim SO, Chung HJ, Jee MS, Kim BG, Kim KM, Yoon JH, Lee HS, Kim CY, Kim S, Yoo W, Hong SP. Population Genotyping of Hepatitis C Virus by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Analysis of Short DNA Fragments. Clin Chem 2005; 51:1123-31. [PMID: 15890891 DOI: 10.1373/clinchem.2004.047506] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Abstract
Background: Identifying hepatitis C virus (HCV) genotypes has become increasingly important for determining clinical course and the outcome of antiviral therapy. Here we describe the development of restriction fragment mass polymorphism (RFMP) analysis, a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay suitable for high-throughput, sensitive, specific genotyping of multiple HCV species.
Methods: The assay is based on PCR amplification and mass measurement of oligonucleotides containing genotype-specific motifs in the 5′ untranslated region, into which a type IIS restriction endonuclease recognition was introduced by PCR amplification. Enzymatic cleavage of the products led to excision of multiple oligonucleotide fragments representing variable regions whose masses were determined by MALDI-TOF MS.
Results: The RFMP assay identified viral genotypes present at concentrations as low as 0.5% and reliably determined their relative abundance. When sera from 318 patients were analyzed, the RFMP assay exhibited 100% concordance with results obtained by clonal sequencing and identified mixed-genotype infections in 22% of the samples, in addition to several subtype variants.
Conclusions: The RFMP assay has practical advantages over existing methods, including better quantitative detection of mixed populations and detection of genotype variants without need for population-based cloning, enabling reliable viral genotyping in laboratories and efficient study of the relationship between viral genotypes and clinical outcome.
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Affiliation(s)
- Yoon Jun Kim
- Department of Internal Medicine, Liver Research Institute, Seoul National University College of Medicine, Seoul, Korea
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Keating S, Coughlan S, Connell J, Sweeney B, Keenan E. Hepatitis C viral clearance in an intravenous drug-using cohort in the Dublin area. Ir J Med Sci 2005; 174:37-41. [PMID: 15868888 DOI: 10.1007/bf03168517] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
Abstract
BACKGROUND The rate of spontaneous HCV viral clearance is reported as 20-25% but recent data indicate a higher frequency in some cohorts. The rate of spontaneous clearance in intravenous drug users has not been reported in an Irish setting. AIMS To determine the rate of spontaneous hepatitis C viral clearance and genotype in an Irish intravenous drug-using cohort. METHODS Drug users attending five drug treatment clinics in the Dublin were investigated. Data were prospectively recorded from January 1997 to June 2001 and follow-up testing completed in 2003. There were 496 HCV antibody positive patients identified and assessed for HCV RNA clearance. All were HIV and hepatitis B negative, 68.8% were male. RESULTS HCV RNA negativity (viral clearance) was documented in 38% of patients. Viral clearance was 47.4% in females and 34.5% in males (p = 000.6). Clearance was independent of age or duration of intravenous drug use. Viral clearance as defined as two negative consecutive HCV RNA tests, a minimum of one year apart, was sustained in 82.2% at two-year follow-up, giving an overall viral clearance of 31.1%. HCV genotype 1 and 3 were most commonly identified at 48.8% and 48.5% respectively in those with chronic infection. CONCLUSIONS Spontaneous HCV viral clearance occurs at a higher frequency than previously reported. Genotype 1 and 3 are commonest in the patient cohort.
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Affiliation(s)
- S Keating
- The Drug Treatment Centre Board, Trinity Court, Pearse Street, Dublin
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24
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Margraf RL, Page S, Erali M, Wittwer CT. Single-Tube Method for Nucleic Acid Extraction, Amplification, Purification, and Sequencing. Clin Chem 2004; 50:1755-61. [PMID: 15308595 DOI: 10.1373/clinchem.2004.035808] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
AbstractBackground: The hepatitis C virus (HCV) genotype determines patient prognosis and duration of treatment, but sequencing of the gene is lengthy and labor-intensive. We used a commercially available nucleic acid extraction system to develop a single-tube extraction-to-sequencing (STETS) method for HCV genotyping.Methods: HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions.Results: The STETS method generated genotyping-quality sequence for a range of HCV titers from 500 to 6 000 000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths >600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube.Conclusions: A single-tube nucleic acid extraction-to-sequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV.
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Affiliation(s)
- Rebecca L Margraf
- ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
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25
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Anand BS, Velez M. Assessment of correlation between serum titers of hepatitis c virus and severity of liver disease. World J Gastroenterol 2004; 10:2409-11. [PMID: 15285030 PMCID: PMC4576299 DOI: 10.3748/wjg.v10.i16.2409] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: The significance of hepatitis C virus (HCV) serum titers has been examined in several clinical situations. There is much evidence that patients with a lower viral load have better response rates to anti-viral therapy compared to those with higher levels. Moreover, a direct association has been observed between serum titers of HCV and transmission rates of the virus. The aim of the present study was to determine if there was any correlation between HCV viral load and the severity of liver disease.
METHODS: Fifty patients with HCV infection were included in the study. These comprised of 34 subjects with a history of alcohol use and 16 non-alcoholics. Quantitative serum HCV RNA assay was carried out using the branched DNA (bDNA) technique. Linear regression analysis was performed between serum viral titers and liver tests. In addition, for the purpose of comparison, the subjects were divided into two groups: those with low viral titers ( < / = 50 genome mEq/ mL) and high titers ( > 50 mEq/mL).
RESULTS: All subjects were men, with a mean ± SD age of 47 ± 7.8 years. The mean HCV RNA level in the blood was 76.3 × 105± 109.1 genome equivalents/mL. There was no correlation between HCV RNA levels and age of the patients (r = 0.181), and the history or amount (g/d) of alcohol consumption (r = 0.07). Furthermore, no correlation was observed between serum HCV RNA levels and the severity of liver disease as judged by the values of serum albumin (r = 0.175), bilirubin (r = 0.217), ALT (r = 0.06) and AST (r = 0.004) levels. Similarly, no significant difference was observed between patients with low viral titers and high titers with respect to any of the parameters.
CONCLUSION: Our results indicate that the severity of liver disease is independent of serum levels of hepatitis C virus. These findings are important since they have a direct impact on the current debate regarding the role of direct cytopathic effect of hepatitis C virus versus immune-mediated injury in the pathogenesis of HCV-related liver damage.
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Affiliation(s)
- Bhupinder-S Anand
- Digestive Diseases Section, VA Medical Center, 2002 Holcombe Blvd. Houston, Texas 77030, USA.
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Othman SB, Trabelsi A, Monnet A, Bouzgarrou N, Grattard F, Beyou A, Bourlet T, Pozzetto B. Evaluation of a prototype HCV NS5b assay for typing strains of hepatitis C virus isolated from Tunisian haemodialysis patients. J Virol Methods 2004; 119:177-181. [PMID: 15158600 DOI: 10.1016/j.jviromet.2004.03.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2004] [Revised: 02/24/2004] [Accepted: 03/02/2004] [Indexed: 11/28/2022]
Abstract
Hepatitis C virus (HCV) strains isolated from 68 haemodialysis Tunisian patients exhibiting chronic infection were genotyped targeting the NS5b region of the HCV genome using a prototype assay developed by Bayer HealthCare-Diagnostics (TRUGENE NS5b HCV). The overall results were compared to those obtained with another assay of the same company based on sequencing of the 5' non-coding region (TRUGENE HCV 5'NC genotyping kit). All strains could be typed by the 5'NC typing kit, but only 62 (91; 2%) by the NS5b prototype assay. All the 62 strains typed by both methods exhibited the same pattern at the type level: 57 were type 1, 3 were type 2, and 2 were type 4. At the subtype level, eight strains that gave undetermined results by the 5'NC kit were successfully typed by the NS5b kit; eight additional strains exhibited discrepant results. The overall agreement between the two assays was 74.2% at the subtype level. In conclusion, the NS5b region appears to be much more accurate than the 5'NC region to subtype HCV strains, especially in those isolated from patients attending haemodialysis centres where the subtype distribution suggests frequent nosocomial transmissions.
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Affiliation(s)
- S Ben Othman
- Laboratoire des Maladies Transmissibles, Department of Microbiology, Faculty of Pharmacy, Monastir, Hôpital Farhat Hached, Sousse, Tunisia
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Pérez R, Jiménez M, Crespo J, Diago M, Enríquez J, Vaquero P, Solá R, Olcoz JL, Romero M, Salmerón J, Blanco MI, Oña M, Melón S, Rodrigo L. Comparative study of the efficacy of an induction dose of interferon-alpha2b with ribavirin compared with standard combined treatment in naive patients with chronic hepatitis C. J Viral Hepat 2003; 10:437-45. [PMID: 14633177 DOI: 10.1046/j.1365-2893.2003.00461.x] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
The efficacy and secondary effects of an induction dose of interferon-alpha2b (IFN-alpha2b) with ribavirin compared with standard combined treatment in naive patients with chronic hepatitis C infection were compared. A prospective study was undertaken between March 1998 and November 2001 in which 84 Spanish hospitals took part. Six hundred and fourteen naive patients (age range 18-65 years) diagnosed with chronic hepatitis C virus (HCV) infection and without cirrhosis or co-infection by other viruses, were included. Patients were divided into two groups. Group A (n = 304) received induction treatment with a daily dose of 5 MU of IFN-alpha2b for 4 weeks, followed by 5 MU three times a week with ribavirin (1000-1200 mg/day, according to weight) until completing 1 year of treatment. Group B (n = 310) received the standard dose of IFN-alpha2b of 3 MU three times per week for 48 weeks together with ribavirin (1000-1200 mg/day, according to weight). Both groups were completely comparable according to age, gender, body weight, transaminase levels, genotype, viral load and hepatic inflammatory activity (Knodell Index). No control group was included for ethical reasons. Pegylated interferon was not available at the time of the study. Serum baseline samples were collected for the determination of genotype. Samples were also collected at baseline, weeks 4, 12, 24, 48 and 72, in order to detect and quantify HCV-RNA. The efficacy of treatment was evaluated by means of sustained viral response (SVR) characterized by persistent negativity of HCV-RNA at the end of the follow-up period. At week 4, the response to treatment was greater in group A (49.6%) compared with group B (34.5%) (P = 0.0002), and was maintained until week 12 (64.1% compared with 55.8% respectively) (P = 0.03). These differences disappeared at week 24, when group A (69%) was compared with group B (65%) (NS). At week 48, the response rate for group A was 50.6% compared with group B 47.4% (NS), and at week 72, the SVR in group A was 46% compared with 40.3% for group B (NS). The global SVR was 43.1%. On analysing the response to treatment according to genotype and viral load, we found that the induction treatment was slightly superior in patients with genotype 1 and an elevated viral load (>2 x 10(6) copies/ml). They achieved a SVR in group A of 39.1% compared with 25.5% in group B (P < 0.05). However, this slight improvement obtained in group A, was achieved at the expense of a greater percentage of dropouts compared with group B (6.4% vs 2.2%, P < 0.01); a greater rate of side effects (58.5 vs 36.7%, P < 0.05) and also a greater percentage of neutropenia (3.1% vs 0.9%, P < 0.05). The induction treatment presented a better initial response, but this was not maintained at the end of treatment, and did not improve the results obtained with the standard treatment. Although the patients with genotype 1 and elevated viral load had a better response with the induction treatment, this was accompanied by a greater percentage of dropouts and secondary effects. It would be interesting to repeat this type of study in the future, using pegylated interferon.
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Affiliation(s)
- R Pérez
- Gastroenterology Service, University Hospital Central de Asturias, Oviedo, Spain
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Henderson DK. Managing occupational risks for hepatitis C transmission in the health care setting. Clin Microbiol Rev 2003; 16:546-68. [PMID: 12857782 PMCID: PMC164218 DOI: 10.1128/cmr.16.3.546-568.2003] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Hepatitis C virus (HCV) infection is a significant contemporary health problem in the United States and elsewhere. Because it is primarily transmitted via blood, hepatitis C infection presents risks for both nosocomial transmission to patients and occupational spread to health care workers. Recent insights into the pathogenesis, immunopathogenesis, natural history, and treatment of infection caused by this unique flavivirus provide a rationale for the use of new strategies for managing occupational hepatitis C infections when they occur. This article reviews this developing information. Recently published data demonstrate success rates in the treatment of "acute hepatitis C syndrome" that approach 100\%, and although these studies are not directly applicable to all occupational infections, they may provide important clues to optimal management strategies. In addition, the article delineates approaches to the prevention of occupational exposures and also addresses the difficult issue of managing HCV-infected health care providers. The article summarizes currently available data about the nosocomial epidemiology of HCV infection and the magnitude of risk and discusses several alternatives for managing exposure and infection. No evidence supports the use of immediate postexposure prophylaxis with immunoglobulin, immunomodulators, or antiviral agents. Based on the very limited data available, the watchful waiting and preemptive therapy strategies described in detail in this article represent reasonable interim approaches to the complex problem of managing occupational HCV infections, at least until more definitive data are obtained.
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Affiliation(s)
- David K Henderson
- Warren G. Magnuson Clinical Center, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Maryland 20892, USA.
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Nolte FS, Green AM, Fiebelkorn KR, Caliendo AM, Sturchio C, Grunwald A, Healy M. Clinical evaluation of two methods for genotyping hepatitis C virus based on analysis of the 5' noncoding region. J Clin Microbiol 2003; 41:1558-64. [PMID: 12682145 PMCID: PMC153875 DOI: 10.1128/jcm.41.4.1558-1564.2003] [Citation(s) in RCA: 71] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2002] [Revised: 11/09/2002] [Accepted: 01/10/2003] [Indexed: 12/11/2022] Open
Abstract
We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5'NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5' noncoding (5'NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5'NC genotyping results. The sensitivities of the methods were assessed by using the 5'NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5'NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5'NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5'NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5'NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5'NC genotyping methods were comparable and dependent on the amplification test used ( approximately 10(3) IU/ml with the qualitative HCV RNA tests and approximately 10(5) IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5'NC test. In conclusion, the performance characteristics of the 5'NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.
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Affiliation(s)
- Frederick S Nolte
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
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30
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Stramer S. US NAT yield: where are we after 2 years? Transfus Clin Biol 2003. [DOI: 10.1016/s1246-7820(02)00278-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
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31
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Babik JM, Holodniy M. Impact of highly active antiretroviral therapy and immunologic status on hepatitis C virus quasispecies diversity in human immunodeficiency virus/hepatitis C virus-coinfected patients. J Virol 2003; 77:1940-50. [PMID: 12525628 PMCID: PMC140862 DOI: 10.1128/jvi.77.3.1940-1950.2003] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
This study analyzes the effect of highly active antiretroviral therapy (HAART), and thus immunologic status, on hepatitis C virus (HCV) load and quasispecies diversity in patients coinfected with the human immunodeficiency virus (HIV) and HCV. Three cohorts of coinfected patients were analyzed retrospectively over a period of 7 to 10 months: group A was antiretroviral drug naïve at baseline and then on HAART for the remainder of the study, group B did not receive antiretroviral therapy at any point, and group C was on HAART for the entire study. HCV quasispecies diversity was analyzed by sequencing hypervariable region 1. In a longitudinal analysis, there was no significant change from baseline in any immunologic, virologic, or quasispecies parameter in any of the three groups. However, in comparison to groups A and B, group C had significantly higher CD4+- and CD8+-cell counts, a trend toward a higher HCV load, and significantly increased number of HCV clones, entropy, genetic distance, and ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site (Ka/Ks). In addition, CD4+-cell count was positively correlated with HCV load, genetic distance, and Ka. Interestingly, patients infected with HCV genotype 2 or 3 had a significantly higher CD4+-cell count, HCV load, genetic distance, and Ka/Ks than those infected with genotype 1. These results suggest that there is no immediate effect of HAART on HCV but that, with prolonged HAART, immune restoration results in an increase in HCV load and quasispecies diversity.
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Affiliation(s)
- Jennifer M Babik
- Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California 94305, USA
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Chen Z, Weck KE. Hepatitis C virus genotyping: interrogation of the 5' untranslated region cannot accurately distinguish genotypes 1a and 1b. J Clin Microbiol 2002; 40:3127-34. [PMID: 12202542 PMCID: PMC130800 DOI: 10.1128/jcm.40.9.3127-3134.2002] [Citation(s) in RCA: 104] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Although the 5' untranslated region (5' UTR) is the most conserved region of the hepatitis C virus (HCV) genome, it has been suggested that interrogation of this region is sufficient for determination of the HCV genotype. We compared two methods of determination of the HCV genotype: (i) direct sequencing of the DNA of the NS-5b region and (ii) reverse line probe assay (LiPA; INNO-LiPA HCV II; Innogenetics N.V.) of the 5' UTR. There was 100% concordance between the two methods for genotype but only 80% concordance for subtype. A significant percentage of genotype 1a isolates were misclassified by LiPA as genotype 1b. Sequence analysis revealed that the only consistent difference in the 5' UTR for these genotype 1a isolates misclassified as genotype 1b was a single nucleotide (A/G) at position -99 of the HCV genome. All isolates with discordant results analyzed had a G at this position, consistent with LiPA determination of these samples as subtype 1b. However, sequence analysis of 222 nucleotides in the NS-5b region clearly identified all of these isolates as subtype 1a. Population distribution data from the University of Pittsburgh Medical Center of over 200 samples analyzed by sequencing of the NS-5b region and over 1,000 samples analyzed by LiPA also indicated that INNO-LiPA HCV II cannot accurately differentiate HCV genotype 1a isolates from HCV genotype 1b isolates. We provide evidence that the A/G at position -99 represents a sequence polymorphism in the HCV genome that cannot differentiate subtype 1a from subtype 1b isolates. In conclusion, the 5' UTR is not heterogeneous enough for use in determination of the HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.
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Affiliation(s)
- Zhenyu Chen
- Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA
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33
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Abstract
The US blood supply has been tested for human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) using nucleic acid amplification testing (NAT) of pools of small number of samples since early 1999. Since the implementation of NAT under an investigational new drug (IND) application, the results for the yield and false positivity have been remarkably consistent for greater than 2 years of testing even among multiple programmes using two different test methodologies and manufacturers: Gen-Probe/Chiron transcription-mediated amplification (TMA) and Roche polymerase chain reaction. All programmes in the US and Canada use NAT as a criterion for cellular as well as frozen product release. The focus of this paper is to provide an update of the programmes in the US and Canada, provide data in support of p24 antigen replacement by HIV-1 NAT and discuss the projections of residual risk of HIV, HCV and hepatitis B virus (HBV) following NAT and the associated cost/benefit.
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Affiliation(s)
- S L Stramer
- National Testing and Reference Laboratories and Blood Services, American Red Cross, 9315 Gaither Road, Gaithersburg, MD 20877, USA.
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34
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Touceda S, Pereira M, Agulla A. [Prevalence of hepatitis C virus genotypes in the area of El Ferrol (La Coruña, Spain)]. Enferm Infecc Microbiol Clin 2002; 20:200-4. [PMID: 12006256 DOI: 10.1016/s0213-005x(02)72789-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
BACKGROUND To determine the prevalence of hepatitis C virus (HCV) genotypes in the area of El Ferrol, as well as their distribution according to risk factors. METHODS A total of 479 patients with hepatitis C were studied, including 254 with no known risk factors, 161 intravenous drug abusers (IVDA) and 64 with a history of blood transfusions. The presence of HCV RNA was studied by RT-PCR, and a reverse hybridization method (INNO-LiPA) was used for genotyping. RESULTS Genotype distribution was as follows: 1b, 269 (56.2%); 1a, 79 (16.5%); 3a, 59 (12.3%); 4c/4d, 35 (7.3%); 1, 19 (4.0%); 2a/2c, 3 (0.6%); 4, 3 (0.6%); 2b, 2 (0.4%). In 10 patients (2.1%) genotype could not be determined. In patients with no known risk factor, the predominant genotype was 1b, detected in 191 of the 254 patients in this group (75.2%). Distribution of genotypes was more varied in the IVDA group, with the most frequent being 1a in 49 (30.4%) and 3a in 43 (26.7%). In the 64 patients who had received transfusions, 1b was predominant, detected in 54 of 64 patients (84.4%). CONCLUSIONS The predominant HCV genotype in our area is 1b. Differences in genotype distribution were observed in the population groups studied, according to their underlying risk factors.
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Affiliation(s)
- Sonia Touceda
- Servicio de Microbiología. Complejo Hospitalario Arquitecto Marcide Profesor Novoa Santos. El Ferrol. La Coruña. Spain
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35
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Podzorski RP. Molecular testing in the diagnosis and management of hepatitis C virus infection. Arch Pathol Lab Med 2002; 126:285-90. [PMID: 11860301 DOI: 10.5858/2002-126-0285-mtitda] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
OBJECTIVES To review hepatitis C virus (HCV), describe the types of molecular-based tests available for the diagnosis and management of HCV infection, and discuss the appropriate utilization of these tests. DATA SOURCES Current information is presented from the published literature, as well as new information where available. STUDY SELECTION A major cause of posttransfusion and community-acquired non-A, non-B hepatitis worldwide is HCV. Approximately 4 million people in the United States are infected with HCV, resulting in 8000 to 10,000 deaths annually. Because HCV is not readily cultured, in vitro molecular-based tests have been developed for use in the diagnosis and treatment of HCV-infected patients. Molecular tests include qualitative and quantitative nucleic acid amplification tests, branched DNA tests, and HCV genotyping assays. Qualitative HCV nucleic acid amplification tests are used routinely in association with serologic tests to help make a diagnosis of infection with HCV. Quantitative HCV testing and genotyping methods have been found to be valuable tools in the treatment of infected patients. A patient's pretreatment HCV viral load and the rate of virus decline during therapy have been shown to correlate with the likelihood of long-term response to antiviral therapy. Information pertaining to the genotype of HCV infecting patients has been shown to be helpful in making recommendations regarding treatment. Certain genotypes of HCV are much more responsive to therapy, allowing a shorter course of treatment. CONCLUSIONS Molecular tests are valuable tools for use in the diagnosis and treatment of patients infected with HCV.
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Affiliation(s)
- Raymond P Podzorski
- Department of Pathology & Immunology, Wayne State University School of Medicine, Detroit, Mich., USA
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36
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Hwang SJ, Lee SD, Lu RH, Chu CW, Wu JC, Lai ST, Chang FY. Hepatitis C viral genotype influences the clinical outcome of patients with acute posttransfusion hepatitis C. J Med Virol 2001. [PMID: 11596085 DOI: 10.1002/jmv.2064] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Most patients with an acute infection of hepatitis C virus (HCV) will develop chronic hepatitis, and only about 15-20% of the cases will resolve spontaneously. The mechanism for the different outcomes in patients with acute HCV infection remains unclear. HCV genotype has been recognized as an important factor affecting the clinical course and outcome of chronic hepatitis C patients. In order to evaluate the role of HCV genotype in the clinical course and outcome of acute posttransfusion hepatitis C, 67 patients with acute posttransfusion hepatitis C from a prospective study of posttransfusion non-A, non-B hepatitis were enrolled. Thirty-nine patients (58.2%) were HCV genotype 1b. Among the 67 patients with acute posttransfusion hepatitis C, 53 (79.1%) progressed to chronic hepatitis. Significantly more patients with genotype 1b than non-1b genotypes developed chronic hepatitis (89.7% vs. 64.3%; P = 0.019). There was no significant difference in gender, mean age, amount of transfused blood, hepatitis symptoms, jaundice, incubation period, peak serum alanine transaminase, or serum HCV RNA titer between patients with HCV genotype 1b and non-1b infections. Patients who developed chronic hepatitis had a significantly greater incidence of genotype 1b infection (66.0% vs. 28.6%; P = 0.013) and a longer incubation period (7.3 weeks vs. 5.4 weeks; P = 0.052) than patients whose infection was resolved. Patients with a genotype 1b infection that resolved itself spontaneously all had an incubation period of less than 6 weeks. Multivariate logistic regression analysis revealed that genotype 1b and an incubation period > or = 6 weeks were significant predictive factors for the development of chronic hepatitis. Therefore, the HCV genotype can influence the outcome of patients with acute HCV infection.
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Affiliation(s)
- S J Hwang
- Department of Family Medicine, Veterans General Hospital-Taipei, National Yang-Ming University School of Medicine, 201 Shih-Pai Road, Section 2, Taipei, 11217, Taiwan, Republic of China
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37
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Abstract
Serologic assays for diagnosis of hepatitis C infection may yield indeterminate results despite improvements in sensitivity and specificity through second- and third-generation assays. Direct detection of hepatitis C virus (HCV) RNA based on qualitative reverse transcription-polymerase chain reaction or transcription-mediated amplification allows diagnosis in the early stages of acute infection and in patients unable to mount an antibody response. Quantitative HCV RNA assays are useful for selecting appropriate antiviral therapies, but until recently they have lacked comparability between tests. More sensitive qualitative assays should be used for determining duration of treatment or recognizing a sustained virologic response to therapy. Hepatitis C virus genotyping can be performed from a limited sequence analysis of the viral genome by using various techniques. Although newer genotyping methods are relatively practicable and are satisfactory for the discrimination of the majority of genotypes, discrimination between subtypes can be challenging. Serologic typing of HCV lacks sensitivity and specificity compared with molecular-based techniques. Recent advances in serologic assays and nucleic acid detection techniques allow physicians to make accurate diagnoses, and these assays serve as important tools in treatment planning.
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38
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Abstract
Hepatitis C virus, a recently identified member of the family Flaviviridae, is an important cause of chronic viral hepatitis and cirrhosis. There are similarities in the nature of the immune response to this pathogen with immunity in other flavivirus and hepatotropic virus infections, such as hepatitis B. However, the high rate of viral persistence after primary hepatitis C infection, and the observation that neutralizing antibodies are not protective, would suggest that there are a number of important differences between hepatitis C, other flaviviruses, and hepatitis B. The phenomenon of quasispecies evolution and other viral factors have been proposed to contribute to immune evasion by hepatitis C virus. In the face of established persistent infection, virus-specific cytotoxic T lymphocytes may exert some control over viral replication. However, these same effectors may also be responsible for the progressive liver damage characteristic of chronic hepatitis C infection. The nature of protective immunity, including the role of innate immune responses early after hepatitis C exposure, remains to be defined.
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Affiliation(s)
- A J Freeman
- Viral Hepatitis Research, Gastrointestinal and Liver Unit, The Prince of Wales Hospital, Randwick, New South Wales, Australia.
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39
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Mihály I, Telegdy L, Ibrányi E, Lukács A, Rókusz L, Bánkuti E E, Dóczy J. Prevalence, genotype distribution and outcome of hepatitis C infections among the employees of the Hungarian Central Hospital for infectious diseases. J Hosp Infect 2001; 49:239-44. [PMID: 11740870 DOI: 10.1053/jhin.2001.1087] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Blood samples from 477 hospital workers (HWs) at the Central Hospital for Infectious Diseases, Budapest, Hungary were tested for hepatitis C virus (HCV) antibodies by enzyme immunoassay (EIA), and 13 (2.7%) of these were found to be HCV antibody positive. Ten (2.7%) were from nursing/housekeeping staff and three (2.9%) from medical staff. HCV antibody positive HWs were detected in 10 of 17 work places, and the prevalence rates in these departments or units varied between 1.2% and 6.5%. The prevalence increased gradually with increasing age, being 0% in these under 21 years of age and 9.5% in those above 50 years of age. Eleven (85%) of 13 HCV antibody positive HWs had HCV RNA in their sera, four of them intermittently during the follow-up period. HCV genotype 1 was present in two HWs, 1b in six HWs, 3a in one HW and 4 in two HWs. Chronic hepatitis C has developed in six (46%) HCV antibody positive HWs. Although the source of infection through needlestick could only be traced directly in one case, circumstantial evidence indicated that the majority of infections were occupationally acquired, originating from percutaneous or mucocutaneous exposure.
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Affiliation(s)
- I Mihály
- Laboratory for Diagnostic Virology, Central Hospital for Infectious Diseases "St. László", Budapest, Hungary.
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40
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Schröter M, Zöllner B, Schäfer P, Laufs R, Feucht HH. Comparison of three HCV genotyping assays: a serological method as a reliable and inexpensive alternative to PCR based assays. J Clin Virol 2001; 23:57-63. [PMID: 11595584 DOI: 10.1016/s1386-6532(01)00186-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
BACKGROUND Determination of hepatitis C virus (HCV) genotypes and subtypes is of rising clinical importance. In times where also an increasing need for cost effectiveness can be observed, the demand for fast and easy performable assays grows. OBJECTIVES To evaluate and compare different genotyping methods regarding their reliability, practicability, and expense in the daily routine. METHODS Sera of 39 patients infected with different HCV subtypes were examined by a serological genotyping assay (NS-4 IBA), by the widely used INNO-LiPA HCV II, and by a nucleotide sequencing method. RESULTS The tests performed equally well in terms of HCV subtyping and no different results were obtained. However, the serotyping assay provided the results in less than half the time needed by the other two assays. Significant differences were also observed regarding the 'hands on' times and the costs. The technical equipment which was necessary to perform the assays is significantly reduced using the serological assay. CONCLUSION Our study demonstrates that the serological test offers the opportunity to determine HCV genotypes and subtypes reliably, fast, easy, and cost effective.
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Affiliation(s)
- M Schröter
- Institut für Medizinische Mikrobiologie und Immunologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
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41
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Yu ML, Chuang WL, Chen SC, Dai CY, Hou C, Wang JH, Lu SN, Huang JF, Lin ZY, Hsieh MY, Tsai JF, Wang LY, Chang WY. Changing prevalence of hepatitis C virus genotypes: molecular epidemiology and clinical implications in the hepatitis C virus hyperendemic areas and a tertiary referral center in Taiwan. J Med Virol 2001. [PMID: 11505444 DOI: 10.1002/jmv.2001] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
To determine the hepatitis C virus (HCV) genotype distribution in Taiwan and to clarify the relationship between genotype and the pathogenesis of HCV infection, 1,164 subjects positive for serum HCV antibodies and HCV RNA from three HCV hyperendemic areas (Masago, Tzukuan, and Taoyuan) and a tertiary referral center in Taiwan were studied during 1995-1997. HCV genotypes and viral loads were determined using Okamoto's method and branched DNA assay, respectively. Genotype 1b was the most prevalent in Tzukuan (61.9%), Taoyuan (76.9%), and the referral center (47.0%). By contrast, genotype 2a was the major HCV type in Masago (63.5%). Prevalence of genotype 1b positively and that of genotype 2a negatively correlated to age, regardless of study populations (P < 0.01). Based on multivariate analysis, the significant factors associated with the presence of cirrhosis, with or without hepatocellular carcinoma, in chronic hepatitis C patients were genotype 1b and age. In conclusion, these results underline that independent HCV outbreaks continue in HCV hyperendemic areas in Taiwan, concomitant with a changing relative prevalence of HCV genotypes in relation to age. Both the correlation of genotype 1b with age (cohort effect) and intrinsic properties of HCV genotypes are probably responsible for the association between genotype and the pathogenesis of HCV infection.
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Affiliation(s)
- M L Yu
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
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42
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Germer JJ, Zein NN. Advances in the molecular diagnosis of hepatitis C and their clinical implications. Mayo Clin Proc 2001; 76:911-20. [PMID: 11560302 DOI: 10.4065/76.9.911] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
Serologic assays for diagnosis of hepatitis C infection may yield indeterminate results despite improvements in sensitivity and specificity through second- and third-generation assays. Direct detection of hepatitis C virus (HCV) RNA based on qualitative reverse transcription-polymerase chain reaction or transcription-mediated amplification allows diagnosis in the early stages of acute infection and in patients unable to mount an antibody response. Quantitative HCV RNA assays are useful for selecting appropriate antiviral therapies, but until recently they have lacked comparability between tests. More sensitive qualitative assays should be used for determining duration of treatment or recognizing a sustained virologic response to therapy. Hepatitis C virus genotyping can be performed from a limited sequence analysis of the viral genome by using various techniques. Although newer genotyping methods are relatively practicable and are satisfactory for the discrimination of the majority of genotypes, discrimination between subtypes can be challenging. Serologic typing of HCV lacks sensitivity and specificity compared with molecular-based techniques. Recent advances in serologic assays and nucleic acid detection techniques allow physicians to make accurate diagnoses, and these assays serve as important tools in treatment planning.
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Affiliation(s)
- J J Germer
- Division of Clinical Microbiology, Mayo Clinic, Rochester, Minn 55905, USA
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43
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Affiliation(s)
- G M Lauer
- Infectious Disease Division and Partners AIDS Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, USA
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44
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Quarleri JF, Robertson BH, Mathet VL, Feld M, Espínola L, Requeijo MP, Mandó O, Carballal G, Oubiña JR. Genomic and phylogenetic analysis of hepatitis C virus isolates from argentine patients: a six-year retrospective study. J Clin Microbiol 2000; 38:4560-4568. [PMID: 11101596 PMCID: PMC87637 DOI: 10.1128/jcm.38.12.4560-4568.2000] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/1999] [Accepted: 09/07/2000] [Indexed: 12/20/2022] Open
Abstract
Typing of hepatitis C virus (HCV) isolates from Argentine patients was performed by using different methodologies in a population of 243 patients. HCV subtype was assigned based upon restriction fragment length polymorphism (RFLP). HCV RNA genomes obtained from serum samples were classified as belonging to clade 1 (53.5%), 2 (23. 0%), or 3 (8.6%); 14.8% of samples showed HCV mixed infections, more frequently implying different subtypes within the same clade. In addition to RFLP typing, phylogenetic relatedness among sequences from both 5' untranslated region (n = 50) and nonstructural 5B coding region (n = 15) was established.
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Affiliation(s)
- J F Quarleri
- Laboratorio de Hepatitis Virales, Departamento Microbiología, Facultad de Medicina, Universidad de Buenos Aires, Argentina
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45
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Abstract
Exposure to blood-borne pathogens poses a serious risk to health care workers (HCWs). We review the risk and management of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections in HCWs and also discuss current methods for preventing exposures and recommendations for postexposure prophylaxis. In the health care setting, blood-borne pathogen transmission occurs predominantly by percutaneous or mucosal exposure of workers to the blood or body fluids of infected patients. Prospective studies of HCWs have estimated that the average risk for HIV transmission after a percutaneous exposure is approximately 0.3%, the risk of HBV transmission is 6 to 30%, and the risk of HCV transmission is approximately 1.8%. To minimize the risk of blood-borne pathogen transmission from HCWs to patients, all HCWs should adhere to standard precautions, including the appropriate use of hand washing, protective barriers, and care in the use and disposal of needles and other sharp instruments. Employers should have in place a system that includes written protocols for prompt reporting, evaluation, counseling, treatment, and follow-up of occupational exposures that may place a worker at risk of blood-borne pathogen infection. A sustained commitment to the occupational health of all HCWs will ensure maximum protection for HCWs and patients and the availability of optimal medical care for all who need it.
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46
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Hu YW, Balaskas E, Furione M, Yen PH, Kessler G, Scalia V, Chui L, Sher G. Comparison and application of a novel genotyping method, semiautomated primer-specific and mispair extension analysis, and four other genotyping assays for detection of hepatitis C virus mixed-genotype infections. J Clin Microbiol 2000; 38:2807-13. [PMID: 10921931 PMCID: PMC87116 DOI: 10.1128/jcm.38.8.2807-2813.2000] [Citation(s) in RCA: 46] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
To date the true prevalence of hepatitis C virus (HCV) mixed-genotype infections has not been established mainly because currently available methods are not suitable for the detection of mixed genotypes in a viral population. A novel semiautomated genotyping method, primer-specific and mispair extension analysis (S-PSMEA), which is more reliable than other genotyping assays was developed for detection of HCV mixed-genotype infections. A genotype present at levels as low as 0.8% in a defined mix of HCV genotypes was detected, showing a 20-fold increase in sensitivity over that of direct DNA sequencing. A total of 434 HCV isolates were genotyped and analyzed for a comparative study of the accuracy between S-PSMEA and four current genotyping methods. The results showed that viruses in approximately 40% of the samples from this group determined to be infected with mixed genotypes by S-PSMEA were undetected by direct DNA sequencing due to its low sensitivity. Type-specific PCR, line probe assay, and restriction fragment length polymorphism analysis performed poorly, being able to identify only 38.5, 16.1, and 15.4% of mixed-genotype infections, respectively, that were detected by direct DNA sequencing. The prevalence of mixed-genotype infections detected by S-PSMEA was 7.9% (12 of 152 donors) among HCV-infected blood donors, 14.3% (15 of 105) among patients with chronic hepatitis C, and 17.1% (6 of 36) among thalassemia patients who had received multiple transfusions. The data lead us to conclude that HCV mixed-genotype infections are more common than previously estimated and that S-PSMEA may be the method of choice when detection of genotypes present at low levels in mixed-genotype infections is required due to its higher level of sensitivity.
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Affiliation(s)
- Y W Hu
- Canadian Blood Services, Ottawa, Ontario, Canada K1G 4J5.
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47
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Beltrami EM, Williams IT, Shapiro CN, Chamberland ME. Risk and management of blood-borne infections in health care workers. Clin Microbiol Rev 2000; 13:385-407. [PMID: 10885983 PMCID: PMC88939 DOI: 10.1128/cmr.13.3.385] [Citation(s) in RCA: 160] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Exposure to blood-borne pathogens poses a serious risk to health care workers (HCWs). We review the risk and management of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections in HCWs and also discuss current methods for preventing exposures and recommendations for postexposure prophylaxis. In the health care setting, blood-borne pathogen transmission occurs predominantly by percutaneous or mucosal exposure of workers to the blood or body fluids of infected patients. Prospective studies of HCWs have estimated that the average risk for HIV transmission after a percutaneous exposure is approximately 0.3%, the risk of HBV transmission is 6 to 30%, and the risk of HCV transmission is approximately 1.8%. To minimize the risk of blood-borne pathogen transmission from HCWs to patients, all HCWs should adhere to standard precautions, including the appropriate use of hand washing, protective barriers, and care in the use and disposal of needles and other sharp instruments. Employers should have in place a system that includes written protocols for prompt reporting, evaluation, counseling, treatment, and follow-up of occupational exposures that may place a worker at risk of blood-borne pathogen infection. A sustained commitment to the occupational health of all HCWs will ensure maximum protection for HCWs and patients and the availability of optimal medical care for all who need it.
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Affiliation(s)
- E M Beltrami
- HIV Infections Branch, Hospital Infections Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333, USA.
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48
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Freeman AJ, Zekry A, Whybin LR, Harvey CE, van Beek IA, de Kantzow SL, Rawlinson WD, Boughton CR, Robertson PW, Marinos G, Lloyd AR. Hepatitis C prevalence among Australian injecting drug users in the 1970s and profiles of virus genotypes in the 1970s and 1990s. Med J Aust 2000; 172:588-91. [PMID: 10914104 DOI: 10.5694/j.1326-5377.2000.tb124124.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
OBJECTIVES To examine the seroprevalence of hepatitis C virus (HCV) in the Australian injecting drug-using community in the 1970s, and to compare the profile of HCV genotypes with that seen in the 1990s. DESIGN Investigation of stored sera that were collected from injecting drug users in the 1970s and comparison with sera collected in the 1990s. SETTING Inner Sydney, 1974-1975 and 1994-1996. PATIENTS The 1970s group comprised 141 consecutive injecting drug users who attended the Brisbane Street Methadone Clinic. The 1990s group comprised 88 consecutive, injecting drug users of European origin who were HCV antibody-positive and attended a primary healthcare facility (the Kirketon Road Centre). MAIN OUTCOME MEASURES HCV antibody prevalence (1970s); profile of HCV serotypes (1970s and 1990s); and serological evidence of hepatitis A and B. RESULTS 84% of the 1970s group were HCV antibody-positive, of whom 92% were infected with HCV serotype 1 and 1% with serotype 3. In contrast, in the 1990s group, 69% were infected with HCV serotype 1 and 25% with serotype 3. The HCV-positive subjects from the early group were more likely than those from the recent group to have serological evidence of previous HBV infection. CONCLUSIONS The high prevalence of HCV among injecting drug users in the 1970s in Australia confirms an epidemic that has been present for at least 25 years. Over this period, the proportion of HCV genotype 1 infections has decreased and genotype 3 infections have emerged.
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Affiliation(s)
- A J Freeman
- Department of Gastroenterology, Prince of Wales Hospital, Sydney, NSW
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49
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White PA, Li Z, Zhai X, Marinos G, Rawlinson WD. Mixed viral infection identified using heteroduplex mobility analysis (HMA). Virology 2000; 271:382-9. [PMID: 10860891 DOI: 10.1006/viro.2000.0323] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
It is now recognised that mixed viral infection, or infection of an individual with two or more distinct strains of a single viral species, often occurs particularly with RNA viruses. Current methods for detection of mixed infection normally involve genotyping or cloning and DNA sequencing. These methods are not always accurate or sensitive at detecting mixed infection and cannot be used for large numbers of samples. Furthermore subsequent sequence determination of the coinfecting viruses is labour intensive. This paper describes a simple, generic method based upon PCR and heteroduplex mobility analysis (HMA) that can be used to rapidly determine mixed infection with two strains of the same virus. The utility of this method is illustrated with hepatitis C virus (HCV) and TT virus (TTV) as examples. PCR-HMA detected mixed infection in 3 (8%) of 38 sera from intravenous drug users (IVDU) and 28 (30%) of 70 TTV-positive sera from Australia, China, and Vietnam. HMA can also be used to screen recombinant colonies to identify the sequences of the coinfecting viruses. The methods described here could be applied to analyse any PCR product containing two or more divergent sequences, whether derived from viruses, bacteria, or eukaryotic organisms.
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Affiliation(s)
- P A White
- Virology Division, Prince of Wales Hospital, Sydney, New South Wales, Australia
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50
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Abstract
Hepatitis C virus (HCV) has been classified into different genotypes/subtypes with demonstrated clinical implications. Whether there is biological difference between genotypes is unknown. We determined HCV genotype in 120 anti-HCV-positive patients with end-stage renal disease and on haemodialysis, by both serological assay (which showed evidence of previous exposure) and by two molecular assays: restriction fragment length polymorphism (RFLP) and line-probe reverse hybridization (LiPA). In mixing experiments, RFLP and LiPA was able to detect the minor HCV genotypes (if present) in 5-30% and 1-2% of the viral population, respectively. Of the 120 patients studied, genotype-specific antibodies were detected in 50 (42%), and eight patients had reactivities to peptides derived from multiple genotypes (genotypes 1 and 2 and/or 3). Only genotype 1 infection was found by RFLP/LiPA in these eight patients with reactivities to multiple HCV genotypes. One-hundred and five of the 120 (88%) patients were positive for HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR) analysis and 14 were found to have mixed genotype infection. Follow-up serum samples (4-21 months later) were available in five patients (genotype 1a with another genotype/subtype). All five patients had a reduced number of HCV genotypes detected during follow-up; four of the five patients still had detectable genotype 1a, and one patient lost genotype 1a and was positive for genotype 2b only. These data showed that HCV mixed infection can be reliably detected by molecular methods and, in patients with end-stage renal disease and mixed genotype infection, there is a trend that during follow-up, HCV genotype 1 may prevail, or 'take over' the genotype 2 and 3 infection.
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Affiliation(s)
- K P Qian
- Section of Hepatobiliary Diseases, Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, University of Florida, Gainesville, FL 32610, USA
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