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Tung HD, Chen JJ. Genetic history of hepatitis C virus genotype 6 in Taiwan. J Formos Med Assoc 2024; 123:926-933. [PMID: 37996321 DOI: 10.1016/j.jfma.2023.10.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2022] [Revised: 03/09/2023] [Accepted: 10/12/2023] [Indexed: 11/25/2023] Open
Abstract
Unlike hepatitis C virus (HCV) genotype (GT) 6, which is widely circulated in Southeast Asia and South China, GT 6 was not reported in Taiwan until 2006. GT 1b and 2a, also known as global HCV subtypes, have been reported as major GTs circulating in Taiwan. Because of improvement in genotyping kits and sequencing techniques for the subtyping of HCV, an increasing number of GT 6 subtypes have been reported, especially subtype 6a among intravenous drug users with human immunodeficiency virus infection after an outbreak since 2003. Thus, HCV GT 6 infection is regarded to be closely associated with injection drug use. However, recently, we found an unexpectedly high GT 6 prevalence in the general population in Tainan, southern Taiwan. Most of these GT 6 samples belonged to a putative novel subtype closely related to 6g and 6w instead of 6a. Phylogenetic analyses indicated that this putative 6g-related novel subtype and 6w could be indigenous in southern Taiwan for centuries. Southern Taiwan could be the origin of HCV subtype 6w. This finding might change the perspective of HCV epidemiology in Taiwan.
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Affiliation(s)
- Hung-Da Tung
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan
| | - Jyh-Jou Chen
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chi-Mei Medical Center, Liouying, Tainan, Taiwan.
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Chang YP, Huang CB, Su TH, Liu CJ, Tseng TC, Huang SC, Chen PJ, Kao JH, Liu CH. Comparison of diagnostic performance among Abbott RealTime HCV Genotyping II, Abbott HCV Genotype plus RUO, and Roche Cobas HCV Genotyping assays for hepatitis C virus genotyping. J Med Virol 2024; 96:e29686. [PMID: 38767142 DOI: 10.1002/jmv.29686] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2024] [Revised: 05/06/2024] [Accepted: 05/11/2024] [Indexed: 05/22/2024]
Abstract
Comparison of diagnostic accuracy for commercial hepatitis C virus (HCV) genotyping (Abbott RealTime HCV Genotyping II, Roche Cobas Genotyping) and investigational Abbott HCV Genotype plus RUO assays designed to discriminate genotype (GT)-1a, 1b or 6 in cases of ambiguous GT from the Abbott commercial assay remains limited. 743 HCV-viremic samples were subjected to analysis using Abbott and Roche commercial as well as Abbott HCV Genotype plus RUO assays. Next-generation sequencing (NGS) targeting core region was employed as the reference standard. Diagnostic accuracy was reported as the number of participants (percentages) along with 95% confidence intervals (CIs). Using NGS, 741 samples (99.7%) yielded valid genotyping results. The diagnostic accuracies were 97.6% (95% CI: 96.1%-98.5%) and 95.3% (95% CI: 93.4%-96.6%) using Abbott and Roche commercial assays (p = 0.0174). Abbott commercial assay accurately diagnosed HCV GT-6a and 6w, whereas Roche commercial assay accurately diagnosed HCV GT-6a. Both assays demonstrated low accuracies for HCV GT-6b, 6e, 6g, and 6n. Abbott HCV Genotype plus RUO assay discriminated 13 of the 14 samples (92.9%; 95% CI: 64.2%-99.6%) that yielded ambiguous GT. Both assays were capable of diagnosing mixed HCV infections when the minor genotype comprised >8.4% of the viral load. The diagnostic performance of commercial HCV genotyping assays is commendable. Abbott assay demonstrated superior performance compared to Roche assay in diagnosing HCV GT-6. Abbott HCV Genotype plus RUO assay aids in discriminating ambiguous GT. Both commercial assays are proficient in diagnosing mixed HCV infections at a cut-off viral load of 8.4% in minor genotype.
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Affiliation(s)
- Yu-Ping Chang
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Chiuan-Bo Huang
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
| | - Tung-Hung Su
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
| | - Chun-Jen Liu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Tai-Chung Tseng
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
- Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
| | - Shang-Chin Huang
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
- Department of Internal Medicine, National Taiwan University Hospital Bei-Hu Branch, Taipei, Taiwan
| | - Pei-Jer Chen
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Jia-Horng Kao
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
- Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
- Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan
| | - Chen-Hua Liu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan
- Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan
- Department of Internal Medicine, National Taiwan University Hospital, Yun-Lin Branch, Douliou, Taiwan
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Dwivedi M, Dwivedi A, Mukherjee D. An Insight into Hepatitis C Virus: In Search of Promising Drug Targets. Curr Drug Targets 2023; 24:1127-1138. [PMID: 37907492 DOI: 10.2174/0113894501265769231020031857] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Revised: 09/13/2023] [Accepted: 09/21/2023] [Indexed: 11/02/2023]
Abstract
Hepatitis C Virus (HCV) is a global health concern, chronically infecting over 70 million people worldwide. HCV is a bloodborne pathogen that primarily affects the liver, and chronic HCV infection can lead to cirrhosis, liver cancer, and liver failure over time. There is an urgent need for more effective approaches to prevent and treat HCV. This review summarizes current knowledge on the virology, transmission, diagnosis, and management of HCV infection. It also provides an in-depth analysis of HCV proteins as promising targets for antiviral drug and vaccine development. Specific HCV proteins discussed as potential drug targets include the NS5B polymerase, NS3/4A protease, entry receptors like CD81, and core proteins. The implications of HCV proteins as diagnostic and prognostic biomarkers are also explored. Current direct-acting antiviral therapies are effective but have cost, genotype specificity, and resistance limitations. This review aims to synthesize essential information on HCV biology and pathogenesis to inform future research on improved preventive, diagnostic, and therapeutic strategies against this global infectious disease threat.
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Affiliation(s)
- Manish Dwivedi
- Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus, Gomtinagar Extension, Lucknow- 226028, India
| | - Aditya Dwivedi
- Amity Institute of Biotechnology, Amity University Uttar Pradesh, Lucknow Campus, Gomtinagar Extension, Lucknow- 226028, India
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Christensen KT, Pierard F, Beuselinck K, Bonsall D, Bowden R, Lagrou K, Nevens F, Schrooten Y, Simmonds P, Vandamme AM, Van Wijngaerden E, Dierckx T, Cuypers L, Van Laethem K. Full-genome next-generation sequencing of hepatitis C virus to assess the accuracy of genotyping by the commercial assay LiPA and the prevalence of resistance-associated substitutions in a Belgian cohort. J Clin Virol 2022; 155:105252. [PMID: 35981443 DOI: 10.1016/j.jcv.2022.105252] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 07/26/2022] [Accepted: 08/02/2022] [Indexed: 02/07/2023]
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Lapointe HR, Dong W, Dong WWY, Kirkby D, Woods C, Poon AFY, Howe AYM, Harrigan PR, Brumme CJ. Validation of a Genotype-Independent Hepatitis C Virus Near-Whole Genome Sequencing Assay. Viruses 2021; 13:v13091721. [PMID: 34578305 PMCID: PMC8473162 DOI: 10.3390/v13091721] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2021] [Revised: 08/17/2021] [Accepted: 08/26/2021] [Indexed: 11/16/2022] Open
Abstract
Despite the effectiveness of direct-acting antiviral agents in treating hepatitis C virus (HCV), cases of treatment failure have been associated with the emergence of resistance-associated substitutions. To better guide clinical decision-making, we developed and validated a near-whole-genome HCV genotype-independent next-generation sequencing strategy. HCV genotype 1-6 samples from direct-acting antiviral agent treatment-naïve and -treated HCV-infected individuals were included. Viral RNA was extracted using a NucliSens easyMAG and amplified using nested reverse transcription-polymerase chain reaction. Libraries were prepared using Nextera XT and sequenced on the Illumina MiSeq sequencing platform. Data were processed by an in-house pipeline (MiCall). Nucleotide consensus sequences were aligned to reference strain sequences for resistance-associated substitution identification and compared to NS3, NS5a, and NS5b sequence data obtained from a validated in-house assay optimized for HCV genotype 1. Sequencing success rates (defined as achieving >100-fold read coverage) approaching 90% were observed for most genotypes in samples with a viral load >5 log10 IU/mL. This genotype-independent sequencing method resulted in >99.8% nucleotide concordance with the genotype 1-optimized method, and 100% agreement in genotype assignment with paired line probe assay-based genotypes. The assay demonstrated high intra-run repeatability and inter-run reproducibility at detecting substitutions above 2% prevalence. This study highlights the performance of a freely available laboratory and bioinformatic approach for reliable HCV genotyping and resistance-associated substitution detection regardless of genotype.
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Affiliation(s)
- Hope R. Lapointe
- Department of Medicine, Division of Social Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; (H.R.L.); (P.R.H.)
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Weiyan Dong
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Winnie W. Y. Dong
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Don Kirkby
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Conan Woods
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
| | - Art F. Y. Poon
- Department of Pathology and Laboratory Medicine, Western University, London, ON N6A 3K7, Canada;
| | - Anita Y. M. Howe
- British Columbia Centre for Disease Control, Vancouver, BC V5Z 4R4, Canada;
| | - P. Richard Harrigan
- Department of Medicine, Division of Social Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; (H.R.L.); (P.R.H.)
| | - Chanson J. Brumme
- BC Centre for Excellence in HIV/AIDS, Vancouver, BC V6Z 1Y6, Canada; (W.D.); (W.W.Y.D.); (D.K.); (C.W.)
- Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
- Correspondence:
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Huang YC, Huang CF, Liu SF, Liu HY, Yeh ML, Huang CI, Hsieh MH, Dai CY, Chen SC, Yu ML, Chuang WL, Huang JF. The performance of HCV GT plus RUO reagent in determining Hepatitis C virus genotypes in Taiwan. PLoS One 2021; 16:e0246376. [PMID: 33513184 PMCID: PMC7845948 DOI: 10.1371/journal.pone.0246376] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Accepted: 01/15/2021] [Indexed: 11/18/2022] Open
Abstract
BACKGROUND AND AIMS Hepatitis C virus (HCV) genotyping is a pivotal tool for epidemiological investigation, guiding management and antiviral treatment. Challenge existed in identifying subtypes of genotype-1 (G-1) and genotype (GT) of indeterminate. Recently, the Abbott HCV RealTime Genotype Plus RUO assay (HCV GT Plus) has been developed aiming to overcome the limitations. We aimed to evaluate the performance of the assay compared with 5' UTR sequencing in clinical samples. MATERIALS AND METHODS Eligible individuals were treatment chronic hepatitis C patients that were enrolled consecutively in a medical center and two core regional hospitals in southern Taiwan from Oct 2017 through Aug 2018. The patient with genotype 1 without subtype and indeterminate previously genotyped by Abbott RealTime HCV GT II will further determinate by Abbott HCV RealTime HCV GT Plus. All of the genotype results were validated by 5' UTR sequencing as a reference standard. RESULTS A total of 100 viremic CHC patients were recruited, including 63 G-1 patients (male: 28), and 37 patients (male: 15) of indeterminate genotyped by Abbott RealTime HCV GT II assay (HCV GT II), respectively. The detection rate of 63 GT1 samples without subtype were 93.7% (59/63), 37 indeterminate samples without genotype were 62.2 (23/37) by HCV GT Plus. 5' UTR sequencing confirmed HCV GT Plus characterized results for 84.7% (50/59) of type1, with 100% (4/4), 82.8 (24/29) and 84.6% (22/26) for 1a, 1b and type6; 65.2% (15/23) of indeterminate with 100% (3/3) and 60% (12/20) for 1b and type 6 samples, respectively. CONCLUSIONS The Abbott RealTime HCV GT Plus RUO assay provides additional performance in GT detection.
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Affiliation(s)
- Ying-Chou Huang
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Chung-Feng Huang
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Shu-Fen Liu
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Hung-Yin Liu
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Ming-Lun Yeh
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Ching-I Huang
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Meng-Hsuan Hsieh
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Chia-Yen Dai
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Shinn-Chern Chen
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Ming-Lung Yu
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Wan-Long Chuang
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
| | - Jee-Fu Huang
- Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Faculty of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Hepatitis Centre, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
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Cuypers L, Thijssen M, Shakibzadeh A, Sabahi F, Ravanshad M, Pourkarim MR. Next-generation sequencing for the clinical management of hepatitis C virus infections: does one test fits all purposes? Crit Rev Clin Lab Sci 2019; 56:420-434. [PMID: 31317801 DOI: 10.1080/10408363.2019.1637394] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
While the prospect of viral cure is higher than ever for individuals infected with the hepatitis C virus (HCV) due to ground-breaking progress in antiviral treatment, success rates are still negatively influenced by HCV's high genetic variability. This genetic diversity is represented in the circulation of various genotypes and subtypes, mixed infections, recombinant forms and the presence of numerous drug resistant variants among infected individuals. Common misclassifications by commercial genotyping assays in combination with the limitations of currently used targeted population sequencing approaches have encouraged researchers to exploit alternative methods for the clinical management of HCV infections. Next-generation sequencing (NGS), a revolutionary and powerful tool with a variety of applications in clinical virology, can characterize viral diversity and depict viral dynamics in an ultra-wide and ultra-deep manner. The level of detail it provides makes it the method of choice for the diagnosis and clinical assessment of HCV infections. The sequence library provided by NGS is of a higher magnitude and sensitivity than data generated by conventional methods. Therefore, these technologies are helpful to guide clinical practice and at the same time highly valuable for epidemiological studies. The decreasing costs of NGS to determine genotypes, mixed infections, recombinant strains and drug resistant variants will soon make it feasible to employ NGS in clinical laboratories, to assist in the daily care of patients with HCV.
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Affiliation(s)
- Lize Cuypers
- Laboratory of Clinical and Epidemiological Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven , Leuven , Belgium
| | - Marijn Thijssen
- Laboratory of Clinical and Epidemiological Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven , Leuven , Belgium
| | - Arash Shakibzadeh
- Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University , Tehran , Iran
| | - Farzaneh Sabahi
- Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University , Tehran , Iran
| | - Mehrdad Ravanshad
- Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University , Tehran , Iran
| | - Mahmoud Reza Pourkarim
- Laboratory of Clinical and Epidemiological Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven , Leuven , Belgium.,Health Policy Research Center, Institute of Health, Shiraz University of Medical Sciences , Shiraz , Iran.,Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine , Tehran , Iran
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Hanafy AS, Soliman S, Abd-Elsalam S. Rescue therapy for chronic hepatitis C virus infection after repeated treatment failures: Impact on disease progression and risk of hepatocellular carcinoma. Hepatol Res 2019; 49:377-384. [PMID: 30570817 DOI: 10.1111/hepr.13303] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/09/2018] [Revised: 12/11/2018] [Accepted: 12/17/2018] [Indexed: 12/14/2022]
Abstract
AIM Retreatment after previous failure of direct-acting antivirals for hepatitis C virus (HCV) infection is a challenging decision. The importance of achieving viral eradication on modification of disease progression and outcome, including the risk of hepatocellular carcinoma (HCC), remains a critical issue to be evaluated. METHODS One hundred patients with repeated failure of sofosbuvir and ribavirin or triple therapy with sofosbuvir, ribavirin, and daclatasvir were divided into a study group (n = 50) given rescue therapy (sofosbuvir, daclatasvir, simeprevir, and ribavirin) or a control group (n = 50) matched for age, sex, and pretreatment variables (Child-Turcotte-Pugh score and Fibrosis-4 score). Follow-up was undertaken after the last non-response to detect serious adverse events, such as hepatic decompensation and development of HCC. RESULTS The study group achieved sustained virologic response (SVR) in 47 of 50 (94%) patients. The control group had significantly higher HCC rates than the study group (7 vs. 1 patients), with an odds ratio of 5.44. The rescue therapy was associated with significantly longer time to the occurrence of adverse events. Repeated treatment failures were associated with progression of FibroScan values in the control group (21 ± 4.5 vs. 10 ± 1.5 kPa, P = 0.001); achieving SVR in the study group stopped fibrosis progression despite non-significant increase from baseline (13.2 ± 3.2 vs. 10.6 ± 0.6, P = 0.12). CONCLUSIONS Rescue treatment for HCV infection was highly effective in achieving SVR, less expensive than the newer agents, and is associated with diminished risk of serious adverse events, mainly HCC in these patients.
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Affiliation(s)
| | - Shaimaa Soliman
- Department of Public Health and Community Medicine, Menofia University, Menofia, Egypt
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Management of HCV-related decompensated cirrhosis with direct-acting antiviral agents: who should be treated? Hepatol Int 2019; 13:165-172. [PMID: 30758786 DOI: 10.1007/s12072-019-09933-8] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/04/2018] [Accepted: 01/14/2019] [Indexed: 12/17/2022]
Abstract
BACKGROUND Medical treatment of decompensated cirrhosis due to hepatitis C virus (HCV) remains a clinical challenge even in the era of direct-acting antiviral drugs (DAAs). We evaluated the efficacy and safety of DAAs in the management of HCV genotype 4-related decompensated cirrhosis. METHODS The study included a treatment group (n = 160) composed of HCV patients with decompensated cirrhosis who received DAAs for 3 months and a matched control group (n = 80) who preferred not to receive DAAs, follow-up was for 24-31 months. RESULTS In treatment group; there were improvements in platelet count, albumin, CTP (p = 0.001) and MELD scores (p = 0.03), a significant reduction in the frequency of hepatic encephalopathy (HE). SVR was achieved in 90%. Hepatocellular carcinoma (HCC) developed in 10% (n = 18) within 6.8 ± 2.5 months after DAAs, survival was higher in the treated vs. the control group (28.9 ± 0.95 vs. 11.4 ± 2.2 months, p = 0.001). Liver volume by ultrasound at a cutoff 495 ml was predictive of complications after DAAs therapy mainly HCC and reduced survival with sensitivity 93.2%, specificity 72%. CONCLUSION HCV with decompensated cirrhosis and adequate liver volume had a 90% SVR with improved CTP&MELD and survival. CLINICAL TRIAL (NCT03547895).
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Abstract
INTRODUCTION Hepatitis C virus (HCV) is divided into 7 genotypes and 67 subtypes. HCV genotype studies reflect the viral transmission patterns as well as human migration routes. In a clinical setting, HCV genotype is a baseline predictor for the sustained virological response (SVR) in chronic hepatitis C patients treated with peginterferon or some direct acting antivirals (DAAs). The Versant HCV genotype 2.0 assay has been globally used for HCV genotyping over a decade. Areas covered: The assay is based on reverse hybridization principle. It is evolved from its former versions, and the accuracy and successful genotyping/subtyping rate are substantially improved. It shows an accuracy of 99-100% for genotypes 1-6. It can also reliably identify subtypes 1a and 1b. However, the assay does not allow a high resolution for many other subtypes. Reasons for indeterminate or inaccurate genotyping/subtyping results are discussed. Expert commentary: Genotyping helps to find the most efficacious and cost-effective treatment regimen. The rapid development of anti-HCV treatment regimens, however, is greatly simplifying laboratory tests. In the near future, the need for HCV genotyping and frequent serial on-treatment HCV RNA tests will decrease along with the wide use of the more potent and pan-genotypic DAA regimens.
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Affiliation(s)
- Ruifeng Yang
- a Peking University People's Hospital, Peking University Hepatology Institute , Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases , Beijing , China
| | - Lai Wei
- a Peking University People's Hospital, Peking University Hepatology Institute , Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases , Beijing , China
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11
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Nolte FS. Molecular Microbiology. PRINCIPLES AND APPLICATIONS OF MOLECULAR DIAGNOSTICS 2018. [PMCID: PMC7150357 DOI: 10.1016/b978-0-12-816061-9.00005-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Background Nucleic acid (NA) amplification techniques are now commonly used to diagnose and manage patients with infectious diseases. The growth in the number of Food and Drug Administration–approved test kits and analyte-specific reagents has facilitated the use of this technology in clinical laboratories. Technological advances in NA amplification techniques, automation, NA sequencing, and multiplex analysis have reinvigorated the field and created new opportunities for growth. Simple, sample-in, answer-out molecular test systems are now widely available that can be deployed in a variety of laboratory and clinical settings. Molecular microbiology remains the leading area in molecular pathology in terms of both the numbers of tests performed and clinical relevance. NA-based tests have reduced the dependency of the clinical microbiology laboratory on more traditional antigen detection and culture methods and created new opportunities for the laboratory to impact patient care. Content This chapter reviews NA testing as it applies to specific pathogens or infectious disease syndromes, with a focus on those diseases for which NA testing is now considered the standard of care and highlights the unique challenges and opportunities that these tests present for clinical laboratories.
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Grebely J, Applegate TL, Cunningham P, Feld JJ. Hepatitis C point-of-care diagnostics: in search of a single visit diagnosis. Expert Rev Mol Diagn 2017; 17:1109-1115. [DOI: 10.1080/14737159.2017.1400385] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Affiliation(s)
- Jason Grebely
- Viral Hepatitis Clinical Research Program, The Kirby Institute, UNSW Sydney, Sydney, Australia
| | - Tanya L. Applegate
- Viral Hepatitis Clinical Research Program, The Kirby Institute, UNSW Sydney, Sydney, Australia
| | - Philip Cunningham
- St Vincent’s Centre for Applied Medical Research, Darlinghurst, Sydney, Australia
| | - Jordan J. Feld
- Toronto Centre for Liver Disease, Sandra Rotman Centre for Global Health, University of Toronto, Toronto, Canada
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Dirani G, Paesini E, Mascetra E, Farabegoli P, Dalmo B, Bartolini B, Garbuglia AR, Capobianchi MR, Sambri V. A novel next generation sequencing assay as an alternative to currently available methods for hepatitis C virus genotyping. J Virol Methods 2017; 251:88-91. [PMID: 29045810 DOI: 10.1016/j.jviromet.2017.10.005] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2017] [Revised: 08/17/2017] [Accepted: 10/05/2017] [Indexed: 12/31/2022]
Abstract
Chronic HCV infection is one of the leading causes of liver-related death and in many countries it is a primary reason for having a liver transplant. HCV genotype identification has long been used in the clinical practice, since different genotypes have different response rates and required different doses and durations of IFN/RBV treatment; moreover both the frequency and the pattern of resistance to different Direct-Acting Antivirals (DAAs) classes are subtype specific. Hence the necessity to make an accurate HCV subtyping becomes a fundamental tool to optimize current and future clinical management of HCV infected subjects. In the present study the performance of a next generation sequencing (NGS: based on the Ion Torrent Platform-Vela Sentosa SQ 301 sequencer) HCV genotyping assay has been evaluated. The current method targets a region of the NS5B gene and it is the unique NGS based market CE-IVD assay. As a comparative method a commercial method based on the detection via reverse hybridization of 5'UTR and core regions (Versant HCV Genotype 2.0 Assay, LiPA, Siemens) was selected. A total 207 plasma samples from HCV infected individuals were used. No selection was made for these samples that were submitted for routine HCV genotyping. The results show Vela NGS assay assigns major number of HCV subtypes with respect LiPA. Concerning genotype 1 and 3, the discrepancy of assigned subtypes for LiPA with respect to Vela NGS assay is not relevant (1.8% and 2%, respectively); in contrast, the difference of assigned subtypes for genotypes 2 and 4 is very high (96.6% and 100%, respectively). The resistance mutations data, except for 1a and 1b subtypes, remain scarce; the future relevant challenge will be to identify subtypes-specific drug resistance mutations, which are essential to create highly personalized therapeutic pathways.
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Affiliation(s)
- G Dirani
- Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina, Cesena, Italy.
| | - E Paesini
- Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina, Cesena, Italy
| | - E Mascetra
- Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina, Cesena, Italy
| | - P Farabegoli
- Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina, Cesena, Italy
| | - B Dalmo
- Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina, Cesena, Italy
| | - B Bartolini
- Virology Laboratory, National Institute for Infectious Diseases "L.Spallanzani", Rome, Italy
| | - A R Garbuglia
- Virology Laboratory, National Institute for Infectious Diseases "L.Spallanzani", Rome, Italy
| | - M R Capobianchi
- Virology Laboratory, National Institute for Infectious Diseases "L.Spallanzani", Rome, Italy
| | - V Sambri
- Unit of Microbiology, The Great Romagna Hub Laboratory, Pievesestina, Cesena, Italy; DIMES, University of Bologna, Bologna, Italy
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14
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Manee N, Thongbaiphet N, Pasomsub E, Chantratita W. Clinical evaluation of a newly developed automated massively parallel sequencing assay for hepatitis C virus genotyping and detection of resistance-association variants. Comparison with a line probe assay. J Virol Methods 2017; 249:31-37. [PMID: 28851606 DOI: 10.1016/j.jviromet.2017.08.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Revised: 07/13/2017] [Accepted: 08/21/2017] [Indexed: 02/08/2023]
Abstract
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. Recently, HCV was classified into 6 major genotypes (GTs) and 67 subtypes (STs). Efficient genotyping has become an essential tool for prognosis and indicating suitable treatment, prior to starting therapy in all HCV-infected individuals. The widely used genotyping assays have limitation with regard to genotype accuracy. This study was a comparative evaluation of exact HCV genotyping in a newly developed automated-massively parallel sequencing (MPS) system, versus the established Line probe assay 2.0 (LiPA), substantiated by Sanger sequencing, using 120 previously identified-HCV RNA positive specimens. LiPA gave identical genotypes in the majority of samples tested with MPS. However, as much as 25% of LiPA did not identify subtypes, whereas MPS did, and 0.83% of results were incompatible. Interestingly, only MPS could identify mixed infections in the remaining cases (1.67%). In addition, MPS could detect Resistance-Associated Variants (RAVs) simultaneously in GT1 in 56.82% of the specimens, which were known to affect drug resistance in the HCV NS3/NS4A and NS5A genomic regions. MPS can thus be deemed beneficial for guiding decisions on HCV therapy and saving costs in the long term when compared to traditional methods.
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Affiliation(s)
- Narathon Manee
- Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
| | - Nipa Thongbaiphet
- Virology Laboratory and Center for Medical Genomics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
| | - Ekawat Pasomsub
- Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; Virology Laboratory and Center for Medical Genomics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
| | - Wasun Chantratita
- Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; Virology Laboratory and Center for Medical Genomics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand.
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15
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Welzel TM, Bhardwaj N, Hedskog C, Chodavarapu K, Camus G, McNally J, Brainard D, Miller MD, Mo H, Svarovskaia E, Jacobson I, Zeuzem S, Agarwal K. Global epidemiology of HCV subtypes and resistance-associated substitutions evaluated by sequencing-based subtype analyses. J Hepatol 2017; 67:224-236. [PMID: 28343981 DOI: 10.1016/j.jhep.2017.03.014] [Citation(s) in RCA: 93] [Impact Index Per Article: 11.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Revised: 02/10/2017] [Accepted: 03/06/2017] [Indexed: 01/22/2023]
Abstract
BACKGROUND & AIMS HCV genotype, subtype, and presence of resistance-associated substitutions (RASs) are key determinants for the selection of direct-acting antiviral (DAA) treatment regimens. However, current HCV genotyping assays have limitations in differentiating between HCV subtypes, and RAS prevalence is largely undefined. The aim of this study was to investigate HCV epidemiology in 12,615 patient samples from 28 different countries across five geographic regions. METHODS We compared HCV genotype and subtypes using INNO-LiPA 2.0 vs. amplicon sequencing among 8,945 patients from phase II/III clinical trials of DAAs. Global HCV molecular epidemiology in 12,615 patients was investigated. Subtype RAS prevalence was determined by population or deep sequencing, and phylogenetic analyses investigating subtype diversity were performed. RESULTS Although there was high concordance between INNO-LiPA and sequencing for genotype determination, INNO-LiPA was insufficient for subtype determination for genotype 2, 3, 4, and 6. Sequencing provided subtype refinement for 42%, 10%, 81%, and 78% of genotype 2, 3, 4, and six patients, respectively. Genotype discordance (genotype 2-genotype 1) was observed in 28 of 950 (3%) genotype 2 patients, consistent with inter-genotype recombinants. Sequencing-based analyses demonstrated variations in regional subtype prevalence, notably within genotype 2, 4 and 6. RAS prevalence varied by subtype, with the clinically relevant NS3 RAS Q80K found in genotype 1a, 5a and 6a and the NS5A RAS Y93H in genotype 1b, 3a, 4b, 4r and 7. CONCLUSIONS Together, these analyses provide an understanding of subtyping accuracy and RAS distribution that are crucial for the implementation of global HCV treatment strategies. LAY SUMMARY Hepatitis C virus (HCV) is highly variable, with seven genotypes and 67 subtypes characterized to date. The aim of this study was to i) compare two different methods of discriminating between genotypes; ii) investigate the prevalence of HCV subtypes for each genotype around the world; iii) find the prevalence of resistance-associated substitutions (RASs) in different subtypes. We found that both methods showed high concordance in genotype discrimination, but specific subtypes were not always identified accurately. Sequencing-based analyses demonstrated variations in regional subtype prevalence for some genotypes, notably within GT2, 4 and 6. RAS prevalence also varied by subtype. These variations could determine how successful different drugs are for treating HCV.
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Affiliation(s)
- Tania M Welzel
- Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany
| | | | | | | | | | | | | | | | | | | | - Ira Jacobson
- Mount Sinai Beth Israel Medical Center, New York, USA
| | - Stefan Zeuzem
- Johann Wolfgang Goethe University Medical Center, Frankfurt am Main, Germany
| | - Kosh Agarwal
- King's College Hospital Foundation Trust, London, UK
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16
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Han MS, Park Y, Kim HS. Comparison of Abbott RealTime genotype II, GeneMatrix restriction fragment mass polymorphism and Sysmex HISCL HCV Gr assays for hepatitis C virus genotyping. ACTA ACUST UNITED AC 2017; 55:1122-1128. [DOI: 10.1515/cclm-2016-0130] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2016] [Accepted: 11/29/2016] [Indexed: 12/13/2022]
Abstract
AbstractBackground:Hepatitis C virus (HCV) genotype is a predictive marker for treatment response. We sequentially evaluated the performances of two nucleic acid amplification tests (NAATs) and one serology assay for HCV genotype: Abbott RealTimegenotype II (RealTimeII), GeneMatrix restriction fragment mass polymorphism (RFMP), and Sysmex HISCL HCV Gr (HISCL Gr).Methods:We examined 281 clinical samples with three assays. The accuracy was assessed using the HCV Genotype Performance Panel PHW204 (SeraCare Life Sciences) for two NAATs. Discrepant cases were re-genotyped by the Versant HCV v.2.0 (line probe 2.0) assay.Results:With the RealTimeII assay, clinic samples were analyzed as follows: genotypes 1b (43.1%), 2 (40.2%), 1 subtypes other than 1a and 1b (12.5%), 3 (1.8%), 4 (1.4%), 1a (0.7%), 6 (0.4%), and mixed (1.1%). The RealTimeII and RFMP assays showed a type concordance rate of 97.5% (274/281) (κ=0.80) and no significant discordance (p=0.25). Both assays accurately genotyped all samples in the Performance Panel by the subtype level. The HISCL Gr assay showed concordance rates of about 91% (κ<0.40) and statistically significant discordances with two NAATs (p<0.05). In confirmation tests, the results of RFMP assay were the most consistent with those of Versant 2.0 assay.Conclusions:The three HCV assays provided genotyping and serotyping results with good concordance rates. The two NAATs (RealTimeII and RFMP) showed comparable performance and good agreement. However, the results of the HISCL Gr assay showed statistically significant differences with those of the NAATs.
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17
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Chronic Hepatitis B, C, and D. Microbiol Spectr 2017; 4. [PMID: 27726758 DOI: 10.1128/microbiolspec.dmih2-0025-2015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Chronic hepatitis B, C, and D virus infections contribute significantly to the morbidity and mortality of immunocompromised individuals. To contextualize discussion of these infections in immunocompromised patients, this paper provides an overview of aspects of infection in normal hosts. It then describes differences in disease, diagnostic testing, and therapeutic management observed in immunocompromised patients.
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18
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El Raziky M, Zayed NA, Abdel Baki A, Mansour SA, Shahin RMH. ITPA gene polymorphism (94C>A) effects on ribavirin-induced anemia during therapy in Egyptian patients with chronic hepatitis C. J Med Virol 2017; 89:1823-1829. [PMID: 28480960 DOI: 10.1002/jmv.24844] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Accepted: 04/11/2017] [Indexed: 12/13/2022]
Abstract
Inosine triphosphatase (ITPA) gene variants can protect against ribavirin (RBV)-induced anemia in patients treated for chronic hepatitis C. The aim of this study was to determine the relationship between genetic variants of ITPA polymorphism, anemia, RBV dose reduction, and treatment response in hepatitis C virus (HCV)-infected patients. This study was conducted on 97 Egyptian chronic HCV patients who were scheduled for pegylated-interferon (PEG-INF) /RBV therapy. ITPA genotypes rs1127354 were determined by Real Time PCR melting curve analysis. Effects of ITPA polymorphism on hemoglobin (Hb) levels, RBV dose reduction and treatment response were analyzed. The homozygous wild genotype (CC) was associated with Hb reduction at week 4 (P = 0.004). The minor allele protected against Hb reduction. No association with sustained virological response was observed (P = 0.492). Female gender; lower baseline Hb and higher baseline WBC were associated with week 4 anemia (P = 0.04; P = 0.023; 0.033, respectively). The ITPA gene polymorphism rs1127354 heterozygous genotype (CA) may influence Hb levels and protect against hemolytic anemia during RBV-containing regimens for HCV. However, such findings were not significantly related to treatment outcomes. Patients with wild ITPA genotype (CC) experienced a more Hb drop and RBV dose reductions more frequently.
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Affiliation(s)
- Maissa El Raziky
- Department of Endemic Medicine and Hepatology, Kasr Al Ainy Hospital, School of Medicine, Cairo University, Cairo, Egypt
| | - Naglaa A Zayed
- Department of Endemic Medicine and Hepatology, Kasr Al Ainy Hospital, School of Medicine, Cairo University, Cairo, Egypt
| | - Amin Abdel Baki
- Department of Tropical Medicine, National Hepatology and Tropical Medicine Research Institute (NHTMRI), Cairo, Egypt
| | - Shimaa A Mansour
- Department of Tropical Medicine, National Hepatology and Tropical Medicine Research Institute (NHTMRI), Cairo, Egypt
| | - Rasha M H Shahin
- Department of Clinical Pathology, Kasr Al Ainy, School of Medicine, Cairo University, Cairo, Egypt
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19
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Olmstead AD, Lee TD, Chow R, Gunadasa K, Auk B, Krajden M, Jassem AN. Development and validation of a real-time, reverse transcription PCR assay for rapid and low-cost genotyping of hepatitis C virus genotypes 1a, 1b, 2, and 3a. J Virol Methods 2017; 244:17-22. [PMID: 28219761 DOI: 10.1016/j.jviromet.2017.02.009] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Revised: 02/10/2017] [Accepted: 02/10/2017] [Indexed: 12/17/2022]
Abstract
Hepatitis C virus (HCV) infection affects millions of people and leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Treatment regimen selection requires HCV genotype (Gt) and Gt 1 subtype determination. Use of a laboratory developed, reverse transcription (RT)-PCR assay was explored as a low-cost, high-throughput screening approach for the major HCV genotypes and subtypes in North America. A commercial line probe assay (LiPA) was used for comparison. Sequencing and/or an alternative PCR assay were used for discordant analyses. Testing of 155 clinical samples revealed that a paired, duplex real-time RT-PCR assay that targets Gts 1a and 3a in one reaction and Gts 1b and 2 in another had 95% overall sensitivity and individual Gt sensitivity and specificity of 98-100% and 85-98%, respectively. The RT-PCR assay detected mixed HCV Gts in clinical and spiked samples and no false-positive reactions occurred with rare Gts 3b, 4, 5, or 6. Implementation of the RT-PCR assay, with some reflex LiPA testing, would cost only a small portion of the cost of using LiPA alone, and can also save 1.5h of hands-on time. The use of a laboratory developed RT-PCR assay for HCV genotyping has the potential to reduce cost and labour burdens in high-volume testing settings.
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Affiliation(s)
- Andrea D Olmstead
- University of British Columbia Centre for Disease Control, Vancouver, British Columbia, Canada
| | - Tracy D Lee
- British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada
| | - Ron Chow
- British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada
| | - Kingsley Gunadasa
- British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada
| | - Brian Auk
- British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada
| | - Mel Krajden
- British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
| | - Agatha N Jassem
- British Columbia Centre for Disease Control Public Health Laboratory, Provincial Health Services Authority, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada.
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20
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Nieto-Aponte L, Quer J, Ruiz-Ripa A, Tabernero D, Gonzalez C, Gregori J, Vila M, Asensio M, Garcia-Cehic D, Ruiz G, Chen Q, Ordeig L, Llorens M, Saez M, Esteban JI, Esteban R, Buti M, Pumarola T, Rodriguez-Frias F. Assessment of a Novel Automatic Real-Time PCR Assay on the Cobas 4800 Analyzer as a Screening Platform for Hepatitis C Virus Genotyping in Clinical Practice: Comparison with Massive Sequencing. J Clin Microbiol 2017; 55:504-509. [PMID: 27927921 PMCID: PMC5277520 DOI: 10.1128/jcm.01960-16] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2016] [Accepted: 11/14/2016] [Indexed: 02/06/2023] Open
Abstract
The unequivocal identification of hepatitis C virus (HCV) subtypes 1a/1b and genotypes 2 to 6 is required for optimizing the effectiveness of interferon-free, direct-acting antiviral therapies. We compared the performance of a new real-time HCV genotyping assay used on the Cobas 4800 system (C4800) with that of high-resolution HCV subtyping (HRCS). In total, 502 samples were used, including 184 samples from chronic HCV patients (from routine laboratory activity during April 2016), 5 stored samples with double HCV genotype infections for testing the limitations of the method, and 313 samples from a screening protocol implemented in our hospital (from May to August 2016) based on the new method to further determine its genotyping accuracy. A total of 282 samples, including 171 from April 2016 (the 13 remaining had too low of a viral load for HRCS), 5 selected with double infections, and 106 from screening, were analyzed by both methods, and 220 were analyzed only by the C4800. The C4800 correctly subtyped 125 of 126 1a/1b samples, and the 1 remaining sample was reported as genotype 1. The C4800 correctly genotyped 38 of 45 non-1a/1b samples (classified by HRCS), and it reported the remaining 7 samples as indeterminate. One hundred two of 106 non-1a/1b genotype samples that were identified using the C4800 for screening were confirmed by HRCS. In the 4 remaining samples, 3 were correctly reported as genotype 1 (without defining the subtype) and 1 was reported as indeterminate. None of the samples were misgenotyped. Four of 7 samples with double HCV infections were correctly genotyped by the C4800. Excluding the 5 selected double-infected samples, the C4800 showed 95.7% concordant results for genotyping HCVs 2 to 6 and 1a/1b subtyping, and 99.2% concordance for subtyping 1a/1b single infections in clinical samples. To improve laboratory workflow, we propose using the C4800 as a first-line test for HCV genotyping and 1a/1b classification, followed by transferring non-1a/1b samples to a center where HRCS is available, if further characterization is needed.
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Affiliation(s)
- Leonardo Nieto-Aponte
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Josep Quer
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
| | - Alicia Ruiz-Ripa
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
| | - David Tabernero
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
| | - Carolina Gonzalez
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Josep Gregori
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
- Roche Diagnostics SL, Sant Cugat del Vallès, Spain
| | - Marta Vila
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Miriam Asensio
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Damir Garcia-Cehic
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
| | - Gerardo Ruiz
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Qian Chen
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
| | - Laura Ordeig
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
| | - Meritxell Llorens
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
| | - Montserrat Saez
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Juan I Esteban
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Unit, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Rafael Esteban
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Unit, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Maria Buti
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Liver Unit, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Tomas Pumarola
- Microbiology Department, Hospital Universitari Vall d'Hebron (HUVH), Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
| | - Francisco Rodriguez-Frias
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d'Hebron (HUVH), Universitat Autònoma de Barcelona (UAB), Barcelona, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
- Viral Hepatitis Laboratory, Liver Unit-Internal Medicine, Vall d'Hebron Research Institute (VHIR), Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
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21
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Fevery B, Verbinnen T, Peeters M, Janssen K, Witek J, Jessner W, De Meyer S, Lenz O. Virology analyses of HCV genotype 4 isolates from patients treated with simeprevir and peginterferon/ribavirin in the Phase III RESTORE study. J Viral Hepat 2017; 24:28-36. [PMID: 27696653 DOI: 10.1111/jvh.12614] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/11/2016] [Accepted: 08/05/2016] [Indexed: 01/08/2023]
Abstract
Simeprevir is a hepatitis C virus NS3/4A protease inhibitor. Hepatitis C virus baseline NS3/4A polymorphisms and emerging mutations were characterized in treatment-naїve and treatment-experienced genotype 4-infected patients treated with simeprevir+peginterferon/ribavirin in the RESTORE study. Population sequencing of the NS3/4A region was performed and in vitro simeprevir activity against site-directed mutants or chimeric replicons with patient-derived NS3 protease sequences was assessed in a transient replicon assay. Simeprevir remained active against most (83/91 [91%]) baseline isolates tested in the chimeric replicon assay. Eight baseline isolates reduced simeprevir activity; these carried I132L or D168E substitutions reducing simeprevir median activity by 4.6- and 39-fold, respectively. Six of these eight isolates were from patients achieving sustained virologic response. Baseline NS3 Q80K polymorphism was not observed in the genotype 4-infected patients. Of the 107 simeprevir-treated patients, 37 did not achieve sustained virologic response for any reason. Of the 32 patients who failed treatment and had sequencing information, 28 (88%) had emerging mutations at NS3 positions 80, 122, 155, 156 and/or 168 at time of failure, similar to those in genotype 1. Emerging mutations were mainly D168V and D168E alone or combined with mutations at position 80. In general, isolates obtained at time of failure displayed high-level in vitro resistance to simeprevir (fold change ≥50) in a chimeric replicon assay with a median simeprevir fold change value of 440, consistent with observed mutations. In conclusion, emerging mutations in genotype 4 patients failing simeprevir+peginterferon/ribavirin treatment were similar to those in genotype 1 and conferred high-level resistance to simeprevir.
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Affiliation(s)
- B Fevery
- Janssen Pharmaceutica NV, Beerse, Belgium
| | | | - M Peeters
- Janssen Pharmaceutica NV, Beerse, Belgium
| | - K Janssen
- Janssen Pharmaceutica NV, Beerse, Belgium
| | - J Witek
- Janssen Research & Development LLC, Titusville, NJ, USA
| | - W Jessner
- Janssen Pharmaceutica NV, Beerse, Belgium
| | - S De Meyer
- Janssen Pharmaceutica NV, Beerse, Belgium
| | - O Lenz
- Janssen Pharmaceutica NV, Beerse, Belgium
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22
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Polilli E, Cento V, Restelli U, Ceccherini-Silberstein F, Aragri M, Di Maio VC, Sciacca A, Santoleri F, Fazii P, Costantini A, Perno CF, Parruti G. Consequences of inaccurate hepatitis C virus genotyping on the costs of prescription of direct antiviral agents in an Italian district. CLINICOECONOMICS AND OUTCOMES RESEARCH 2016; 8:467-473. [PMID: 27695353 PMCID: PMC5028103 DOI: 10.2147/ceor.s106238] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Available commercial assays may yield inaccurate hepatitis C virus (HCV) genotype assignment in up to 10% of cases. We investigated the cost-effectiveness of re-evaluating HCV genotype by population sequencing, prior to choosing a direct acting antiviral (DAA) regimen. Between March and September 2015, HCV sequence analysis was performed in order to confirm commercial LiPA-HCV genotype (Versant® HCV Genotype 2.0) in patients eligible for treatment with DAAs. Out of 134 consecutive patients enrolled, sequencing yielded 21 (15.7%) cases of discordant results. For three cases of wrong genotype assignment, the putative reduction in efficacy was gauged between 15% and 40%. Among the eight cases for whom G1b was assigned by commercial assays instead of G1a, potentially suboptimal treatments would have been prescribed. Finally, for five patients with G1 and indeterminate subtype, the choice of regimens would have targeted the worst option, with a remarkable increase in costs, as in the case of the four mixed HCV infections for whom pan-genotypic regimens would have been mandatory. Precise assignment of HCV genotype and subtype by sequencing may, therefore, be more beneficial than expected, until more potent pan-genotypic regimens are available for all patients.
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Affiliation(s)
- Ennio Polilli
- Infectious Diseases Unit, Pescara General Hospital, Pescara
| | - Valeria Cento
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome
| | - Umberto Restelli
- CREMS - Centre for Research on Health Economics, Social and Health Care Management, Carlo Cattaneo - LIUC University, Castellanza, Italy; School of Public Health, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
| | | | - Marianna Aragri
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome
| | - Velia Chiara Di Maio
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome
| | | | | | - Paolo Fazii
- Microbiology and Virology Unit, Pescara General Hospital, Pescara, Italy
| | | | - Carlo Federico Perno
- Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata", Rome
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23
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Cuypers L, Ceccherini-Silberstein F, Van Laethem K, Li G, Vandamme AM, Rockstroh JK. Impact of HCV genotype on treatment regimens and drug resistance: a snapshot in time. Rev Med Virol 2016; 26:408-434. [PMID: 27401933 DOI: 10.1002/rmv.1895] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2016] [Revised: 06/11/2016] [Accepted: 06/15/2016] [Indexed: 12/11/2022]
Abstract
The introduction of highly potent direct-acting antivirals (DAAs) has revolutionized hepatitis C virus treatment. Nevertheless, viral eradication worldwide remains a challenge also in the era of DAA treatment, because of the high associated costs, high numbers of undiagnosed patients, high re-infection rates in some risk groups and suboptimal drug efficacies associated with host and viral factors as well as advanced stages of liver disease. A correct determination of the HCV genotype allows administration of the most appropriate antiviral regimen. Additionally, HCV genetic sequencing improves our understanding of resistance-associated variants, either naturally occurring before treatment, acquired by transmission at HCV infection, or emerging after virological failure. Because treatment response rates, and the prevalence and development of drug resistance variants differ for each DAA regimen and HCV genotype, this review summarizes treatment opportunities per HCV genotype, and focuses on viral genetic sequencing to guide clinical decision making. Although approval of the first pan-genotypic DAA-only regimen is expected soon, HCV genetic sequencing will remain important because when DAA therapies fail, genotyping and resistance testing to select a new active DAA combination will be essential. Copyright © 2016 John Wiley & Sons, Ltd.
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Affiliation(s)
- Lize Cuypers
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium
| | | | - Kristel Van Laethem
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium
| | - Guangdi Li
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium.,Department of Metabolism and Endocrinology, Metabolic Syndrome Research Center, Key Laboratory of Diabetes Immunology, Ministry of Education, National Clinical Research Center for Metabolic Diseases, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
| | - Anne-Mieke Vandamme
- KU Leuven - University of Leuven, Department of Microbiology and Immunology, Rega Institute for Medical Research, Clinical and Epidemiological Virology, Leuven, Belgium.,Center for Global Health and Tropical Medicine, Microbiology Unit, Institute for Hygiene and Tropical Medicine, University Nova de Lisboa, Lisbon, Portugal
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24
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Welker MW, Luhne S, Lange CM, Vermehren J, Farnik H, Herrmann E, Welzel T, Zeuzem S, Sarrazin C. Lactic acidosis in patients with hepatitis C virus cirrhosis and combined ribavirin/sofosbuvir treatment. J Hepatol 2016; 64:790-9. [PMID: 26658684 DOI: 10.1016/j.jhep.2015.11.034] [Citation(s) in RCA: 56] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/08/2015] [Revised: 11/12/2015] [Accepted: 11/23/2015] [Indexed: 12/19/2022]
Abstract
BACKGROUND & AIMS Sofosbuvir (SOF) based interferon-alfa free antiviral therapy has become the treatment of choice for patients with chronic hepatitis C virus (HCV) infection. Little is known about safety of drug combinations using two nucleos(t)ide polymerase inhibitors in patients with HCV associated advanced cirrhosis. Here, we report frequent occurrence of lactic acidosis associated with acute-on-chronic hepatic decompensation during ribavirin (RBV) plus SOF based antiviral therapy. METHODS Thirty-five patients with chronic hepatitis C and advanced fibrosis, compensated cirrhosis, and decompensated cirrhosis without and after liver transplantation were treated with SOF based antiviral therapy with and without RBV. Adverse events including lactic acidosis (pH <7.35, lactate >20 mg/dl) were recorded 24 weeks before and during (mean ±SD, 18±11 weeks) antiviral therapy. Efficacy was determined by assessment of serum HCV RNA. RESULTS We observed severe adverse events in 15/35 (43%) patients before (24 weeks) and in 12/35 (34%) patients during antiviral therapy, the majority in association with acute-on-chronic hepatic decompensation. Lactic acidosis occurred in 5/35 (14%) patients during therapy, while no event of lactic acidosis was observed prior to therapy. Lactic acidosis was associated with hepatic decompensation including renal failure and infection, and was severe (pH <7.3) in two patients. CONCLUSIONS RBV in combination with SOF based antiviral therapy in patients with HCV associated advanced cirrhosis may be associated with the development of lactic acidosis. Impaired renal function, and higher MELD/Child-Pugh scores were identified as potential risk factors.
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Affiliation(s)
- Martin-Walter Welker
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
| | - Stefan Luhne
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Christian M Lange
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Johannes Vermehren
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Harald Farnik
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Eva Herrmann
- Institut für Biostatistik und Mathematische Modellierung, Goethe Universität Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Tania Welzel
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Stefan Zeuzem
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
| | - Christoph Sarrazin
- Medizinische Klinik 1, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
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25
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Molecular Detection and Characterization of Hepatitis C Virus. Mol Microbiol 2016. [DOI: 10.1128/9781555819071.ch31] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Sariguzel FM, Berk E, Gokahmetoglu S, Ercal BD, Celik I. Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping. Pak J Med Sci 2015; 31:1136-9. [PMID: 26649001 PMCID: PMC4641270 DOI: 10.12669/pjms.315.7454] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
Objective: The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey. Methods: One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method. Results: Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b. Conclusion: The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required.
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Affiliation(s)
- Fatma Mutlu Sariguzel
- Fatma Mutlu Sariguzel, Department of Microbiology, Kayseri Education and Research Hospital, Kayseri, Turkey
| | - Elife Berk
- Elife Berk, Department of Microbiology, Erciyes University Medical School, Kayseri, Turkey
| | - Selma Gokahmetoglu
- Selma Gokahmetoglu, Department of Microbiology, Erciyes University Medical School, Kayseri, Turkey
| | - Baris Derya Ercal
- Baris Derya Ercal, Department of Microbiology, Erciyes University Medical School, Kayseri, Turkey
| | - Ilhami Celik
- Ilhami Celik, Department of Infectious Diseases and Clinical Microbiology, Kayseri Education and Research Hospital, Kayseri, Turkey
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27
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Karchava M, Waldenström J, Parker M, Hallack R, Sharvadze L, Gatserelia L, Chkhartishvili N, Dvali N, Dzigua L, Dolmazashvili E, Norder H, Tsertsvadze T. High incidence of the hepatitis C virus recombinant 2k/1b in Georgia: Recommendations for testing and treatment. Hepatol Res 2015; 45:1292-8. [PMID: 25689487 PMCID: PMC4787595 DOI: 10.1111/hepr.12505] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/23/2014] [Revised: 01/23/2015] [Accepted: 02/10/2015] [Indexed: 02/06/2023]
Abstract
AIM The first hepatitis C virus (HCV) recombinant, RF2k/1b, was initially described from Russia and has since then been identified from patients in Ireland, Estonia, Uzbekistan and Cyprus. Many of these patients originated from Georgia; however, there is no information on its prevalence in Georgia or its susceptibility to antiviral treatment. METHODS We retrospectively sequenced the non-structural region 5B (NS5B) of the HCV genome in samples from 72 Georgian patients, 36 of whom had been treated with pegylated interferon and ribavirin. RESULTS The HCV genotype was determined using the Versant HCV Genotype v2 kit. Based on this typing, 32 patients (44.4%) were infected with genotype 1, 21 (29.1%) genotype 2 and 19 (26.3%) genotype 3. Partial NS5B of these strains was sequenced and analyzed for type, with concordant genotype results for all type 1 and 3 strains. Discrepant results were observed for genotyped 2 strains, with 16 (76%) having NS5B of subtype 1b. On phylogenetic analysis, 15 NS5B sequences of these strains were found in a clade formed by recombinant RF2k/1b strains. The remaining discordant sequence was found within a clade formed by 1b strains. CONCLUSION Our findings show that the RF2k/1b recombinant strain is common among Georgian patients previously assumed to be infected with genotype 2. Because genotyping is mainly performed to decide treatment strategies, there is a need to determine the genotype by analysis of at least two genomic regions in strains from Georgian patients considered infected with genotype 2 based on standard HCV genotyping methods.
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Affiliation(s)
- Marine Karchava
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address
| | - Jesper Waldenström
- Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden
| | - Monica Parker
- David Axelrod Institute, Wadsworth Center, Albany, NY, US
| | - Renee Hallack
- David Axelrod Institute, Wadsworth Center, Albany, NY, US
| | - Lali Sharvadze
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address,Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia
| | - Lana Gatserelia
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address
| | - Nikoloz Chkhartishvili
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address
| | - Natia Dvali
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address
| | - Lela Dzigua
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address
| | | | - Helene Norder
- Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden
| | - Tengiz Tsertsvadze
- Infectious Diseases, AIDS and Clinical Immunology Research Center, Tbilisi, Georgia address,Ivane Javakhishvili Tbilisi State University, Tbilisi, Georgia
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28
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Characterization of Samples Identified as Hepatitis C Virus Genotype 1 without Subtype by Abbott RealTime HCV Genotype II Assay Using the New Abbott HCV Genotype Plus RUO Test. J Clin Microbiol 2015; 54:296-9. [PMID: 26582834 PMCID: PMC4733213 DOI: 10.1128/jcm.02264-15] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2015] [Accepted: 11/11/2015] [Indexed: 01/06/2023] Open
Abstract
Hepatitis C virus (HCV) genotyping continues to be relevant for therapeutic strategies. Some samples are reported as genotype 1 (gt 1) without subtype by the Abbott RealTime HCV Genotype II (GT II) test. To characterize such samples further, the Abbott HCV Genotype Plus RUO (Plus) assay, which targets the core region for gt 1a, gt 1b, and gt 6 detection, was evaluated as a reflex test in reference to NS5B or 5′-untranslated region (UTR)/core region sequencing. Of 3,626 routine samples, results of gt 1 without subtype were received for 171 samples (4.7%), accounting for 11.5% of gt 1 specimens. The Plus assay and sequencing were applied to 98 of those samples. NS5B or 5′-UTR/core region sequencing was successful for 91/98 specimens (92.9%). Plus assay and sequencing results were concordant for 87.9% of specimens (80/91 samples). Sequencing confirmed Plus assay results for 82.6%, 85.7%, 100%, and 89.3% of gt 1a, gt 1b, gt 6, and non-gt 1a/1b/6 results, respectively. Notably, 12 gt 6 samples that had been identified previously as gt 1 without subtype were assigned correctly here; for 25/28 samples reported as “not detected” by the Plus assay, sequencing identified the samples as gt 1 with subtypes other than 1a/1b. The genetic variability of HCV continues to present challenges for the current genotyping platforms regardless of the applied methodology. Samples identified by the GT II assay as gt 1 without subtype can be further resolved and reliably characterized by the new Plus assay.
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29
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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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30
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Hepatitis C virus genotype 4 resistance and subtype demographic characterization of patients treated with ombitasvir plus paritaprevir/ritonavir. Antimicrob Agents Chemother 2015; 59:6807-15. [PMID: 26282418 PMCID: PMC4604390 DOI: 10.1128/aac.01229-15] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2015] [Accepted: 08/09/2015] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) genotype 4 (GT4) is genetically diverse, with 17 confirmed subtypes, and comprises approximately 13% of infections worldwide. In this study, we identified GT4 subtypes by phylogenetic analysis, assessed differences in patient demographics across GT4 subtypes, examined baseline sequence variability among subtypes and the potential impact on treatment outcome, and analyzed the development of viral resistance in patients who received a regimen of ombitasvir (nonstructural protein 5A [NS5A] inhibitor) plus ritonavir-boosted paritaprevir (NS3/4A inhibitor) with or without ribavirin (RBV) for the treatment of HCV GT4 infection. Phylogenetic analysis of HCV NS3/4A, NS5A, and NS5B nucleotide sequences identified 7 subtypes (4a, 4b, 4c, 4d, 4f, 4g/4k, and 4o) among 132 patient samples. Subtype prevalence varied by country, and the distributions of patient birth cohort and race were significantly different across GT4 subtypes 4a, 4d, and non-4a/4d. Baseline amino acid variability was detected in NS5A across GT4 subtypes but had no impact on treatment outcome. Three patients experienced virologic failure and were infected with subtype 4d, and the predominant resistance-associated variants at the time of failure were D168V in NS3 and L28V in NS5A. Overall, high response rates were observed among patients infected with 7 HCV GT4 subtypes, with no impact of baseline variants on treatment outcome. GT4 subtype distribution in this study differed based on patient demographics and geography.
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31
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Hajji H, Aherfi S, Motte A, Ravaux I, Mokhtari S, Ruiz JM, Poizot-Martin I, Tourres C, Tivoli N, Gérolami R, Tamalet C, Colson P. Diversity of 1,213 hepatitis C virus NS3 protease sequences from a clinical virology laboratory database in Marseille university hospitals, southeastern France. J Med Virol 2015; 87:1921-33. [PMID: 25959702 DOI: 10.1002/jmv.24261] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/06/2015] [Indexed: 12/21/2022]
Abstract
Infection with hepatitis C virus (HCV) represents a major public health concern worldwide. Recent therapeutic advances have been considerable, HCV genotype continuing to guide therapeutic management. Since 2008, HCV genotyping in our clinical microbiology laboratory at university hospitals of Marseille, Southeastern France, has been based on NS3 protease gene population sequencing, to allow concurrent HCV genotype and protease inhibitor (PI) genotypic resistance determinations. We aimed, first, to analyze the genetic diversity of HCV NS3 protease obtained from blood samples collected between 2003 and 2013 from patients monitored at university hospitals of Marseille and detect possible atypical sequences; and, second, to identify NS3 protease amino acid patterns associated with decreased susceptibility to HCV PIs. A total of 1,213 HCV NS3 protease sequences were available in our laboratory sequence database. We implemented a strategy based on bioinformatic tools to determine whether HCV sequences are representative of our local HCV genetic diversity, or divergent. In our 2003-2012 HCV NS3 protease sequence database, we delineated 32 clusters representative of the majority HCV genetic diversity, and 61 divergent sequences. Five of these divergent sequences showed less than 85% nucleotide identity with their top GenBank hit. In addition, among the 294 sequences obtained in 2013, three were divergent relative to these 32 previously delineated clusters. Finally, we detected both natural and on-treatment genotypic resistance to HCV NS3 PIs, including a substantial prevalence of Q80K substitutions associated with decreased susceptibility to simeprevir, a second generation PI.
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Affiliation(s)
- Hind Hajji
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France
| | - Sarah Aherfi
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France.,Aix-Marseille University, URMITE UM 63 CNRS 7278 IRD 198 INSERM U1905, Facultés de Médecine et de Pharmacie, Marseille, France
| | - Anne Motte
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France
| | - Isabelle Ravaux
- IHU Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Service de Maladies Infectieuses, Centre Hospitalo-Universitaire Conception, Assistance Publique-Hôpitaux de Marseille, Marseille, France
| | - Saadia Mokhtari
- IHU Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Service de Maladies Infectieuses, Centre Hospitalo-Universitaire Nord, Marseille, France
| | - Jean-Marie Ruiz
- Assistance Publique-Hôpitaux de Marseille, Hôpitaux Sud, Service de Médecine en milieu pénitentiaire, Centre pénitentiaire de Marseille, Marseille, France
| | - Isabelle Poizot-Martin
- AP-HM Sainte-Marguerite, Service d'Immuno-hématologie clinique, Marseille, France.,Aix-Marseille University, INSERM, UMR 912 (SESSTIM), Marseille, France
| | - Christian Tourres
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France
| | - Natacha Tivoli
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France
| | - René Gérolami
- Service d'Hépato-Gastro-Entérologie, Centre Hospitalo-Universitaire Conception, Marseille, France
| | - Catherine Tamalet
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France.,Aix-Marseille University, URMITE UM 63 CNRS 7278 IRD 198 INSERM U1905, Facultés de Médecine et de Pharmacie, Marseille, France
| | - Philippe Colson
- Institut Hospitalo-Universitaire (IHU), Méditerranée Infection, Pôle des Maladies Infectieuses et Tropicales Clinique et Biologique, Fédération de Bactériologie-Hygiène-Virologie, Centre Hospitalo-Universitaire Timone, Marseille, France.,Aix-Marseille University, URMITE UM 63 CNRS 7278 IRD 198 INSERM U1905, Facultés de Médecine et de Pharmacie, Marseille, France
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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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33
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Liu CH, Liang CC, Liu CJ, Lin CL, Su TH, Yang HC, Chen PJ, Chen DS, Kao JH. Comparison of Abbott RealTime HCV Genotype II with Versant line probe assay 2.0 for hepatitis C virus genotyping. J Clin Microbiol 2015; 53:1754-1757. [PMID: 25740780 PMCID: PMC4400777 DOI: 10.1128/jcm.03548-14] [Citation(s) in RCA: 63] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2014] [Accepted: 03/01/2015] [Indexed: 11/20/2022] Open
Abstract
Genotyping and subtyping of 225 samples with hepatitis C virus (HCV) genotype 1, 2, 3, or 6 infection were done with Versant LiPA 2.0 and Abbott RealTime HCV Genotype (GT) II by using direct sequencing of the NS5B and 5' untranslated regions as the reference standards. The concordance rates were >99.2% for genotypes and 96.1% for subtypes 1a and 1b. Both the Abbott RealTime and Versant LiPA assays can accurately determine hepatitis C virus genotypes. (This study has been registered at ClinicalTrials.gov under registration no. NCT00979979.).
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Affiliation(s)
- Chen-Hua Liu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan Department of Internal Medicine, National Taiwan University Hospital, Yun-Lin Branch, Douliou, Taiwan
| | - Cheng-Chao Liang
- Department of Internal Medicine, Far Eastern Memorial Hospital, Taipei, Taiwan
| | - Chun-Jen Liu
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Chih-Lin Lin
- Department of Internal Medicine, Taipei Municipal Hospital, Ren-Ai Branch, Taipei, Taiwan
| | - Tung-Hung Su
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Hung-Chih Yang
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan Department of Microbiology, National Taiwan University College of Medicine and National Taiwan University Hospital, Taipei, Taiwan
| | - Pei-Jer Chen
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
| | - Ding-Shinn Chen
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Genomics Research Center, Academia Sinica, Taipei, Taiwan
| | - Jia-Horng Kao
- Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan Hepatitis Research Center, National Taiwan University Hospital, Taipei, Taiwan Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan
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34
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Qiu P, Stevens R, Wei B, Lahser F, Howe AYM, Klappenbach JA, Marton MJ. HCV genotyping from NGS short reads and its application in genotype detection from HCV mixed infected plasma. PLoS One 2015; 10:e0122082. [PMID: 25830316 PMCID: PMC4382110 DOI: 10.1371/journal.pone.0122082] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2014] [Accepted: 02/10/2015] [Indexed: 12/12/2022] Open
Abstract
Genotyping of hepatitis C virus (HCV) plays an important role in the treatment of HCV. As new genotype-specific treatment options become available, it has become increasingly important to have accurate HCV genotype and subtype information to ensure that the most appropriate treatment regimen is selected. Most current genotyping methods are unable to detect mixed genotypes from two or more HCV infections. Next generation sequencing (NGS) allows for rapid and low cost mass sequencing of viral genomes and provides an opportunity to probe the viral population from a single host. In this paper, the possibility of using short NGS reads for direct HCV genotyping without genome assembly was evaluated. We surveyed the publicly-available genetic content of three HCV drug target regions (NS3, NS5A, NS5B) in terms of whether these genes contained genotype-specific regions that could predict genotype. Six genotypes and 38 subtypes were included in this study. An automated phylogenetic analysis based HCV genotyping method was implemented and used to assess different HCV target gene regions. Candidate regions of 250-bp each were found for all three genes that have enough genetic information to predict HCV genotypes/subtypes. Validation using public datasets shows 100% genotyping accuracy. To test whether these 250-bp regions were sufficient to identify mixed genotypes, we developed a random primer-based method to sequence HCV plasma samples containing mixtures of two HCV genotypes in different ratios. We were able to determine the genotypes without ambiguity and to quantify the ratio of the abundances of the mixed genotypes in the samples. These data provide a proof-of-concept that this random primed, NGS-based short-read genotyping approach does not need prior information about the viral population and is capable of detecting mixed viral infection.
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Affiliation(s)
- Ping Qiu
- Molecular Biomarker and Diagnostics, Merck Research Laboratories, Rahway, New Jersey, United States of America
- * E-mail:
| | - Richard Stevens
- Target & Pathway Biology, Merck Research Laboratories, Boston, Massachusetts, United States of America
| | - Bo Wei
- Molecular Biomarker and Diagnostics, Merck Research Laboratories, Rahway, New Jersey, United States of America
| | - Fred Lahser
- Infectious Diseases and Clinical Virology, Merck Research Laboratories, Kenilworth, New Jersey, United States of America
| | - Anita Y. M. Howe
- Infectious Diseases and Clinical Virology, Merck Research Laboratories, Kenilworth, New Jersey, United States of America
| | - Joel A. Klappenbach
- Target & Pathway Biology, Merck Research Laboratories, Boston, Massachusetts, United States of America
| | - Matthew J. Marton
- Molecular Biomarker and Diagnostics, Merck Research Laboratories, Rahway, New Jersey, United States of America
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35
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Murphy DG, Sablon E, Chamberland J, Fournier E, Dandavino R, Tremblay CL. Hepatitis C virus genotype 7, a new genotype originating from central Africa. J Clin Microbiol 2015; 53:967-72. [PMID: 25520447 PMCID: PMC4390628 DOI: 10.1128/jcm.02831-14] [Citation(s) in RCA: 114] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2014] [Accepted: 12/10/2014] [Indexed: 12/18/2022] Open
Abstract
We report a new hepatitis C virus (HCV) genotype identified in patients originating from the Democratic Republic of Congo. The prototype QC69 virus is shown to be a new lineage distinct from genotypes 1 to 6. Three additional patients were also found to be infected by a virus from this lineage, confirming its circulation in humans. We propose that these viruses be classified into HCV genotype 7.
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Affiliation(s)
- Donald G Murphy
- Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Sainte-Anne-de-Bellevue, Québec, Canada
| | | | - Jasmine Chamberland
- Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Sainte-Anne-de-Bellevue, Québec, Canada
| | - Eric Fournier
- Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Sainte-Anne-de-Bellevue, Québec, Canada
| | - Raymond Dandavino
- Département de Néphrologie, Hôpital Maisonneuve-Rosemont, Montréal, Québec, Canada
| | - Cécile L Tremblay
- Laboratoire de Santé Publique du Québec, Institut National de Santé Publique du Québec, Sainte-Anne-de-Bellevue, Québec, Canada
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36
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Firdaus R, Saha K, Biswas A, Sadhukhan PC. Current molecular methods for the detection of hepatitis C virus in high risk group population: A systematic review. World J Virol 2015; 4:25-32. [PMID: 25674515 PMCID: PMC4308525 DOI: 10.5501/wjv.v4.i1.25] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Revised: 11/20/2014] [Accepted: 12/31/2014] [Indexed: 02/05/2023] Open
Abstract
Hepatitis C virus (HCV) is an emerging infection worldwide and the numbers of persons infected are increasing every year. Poor blood transfusion methods along with unsafe injection practices are potential sources for the rapid spread of infection. Early detection of HCV is the need of the hour especially in high risk group population as these individuals are severely immunocompromised. Enzyme Immunoassays are the most common detection techniques but they provide no evidence of active viremia or identification of infected individuals in the antibody-negative phase and their efficacy is limited in individuals within high risk group population. Molecular virological techniques have an important role in detecting active infection with utmost specificity and sensitivity. Technologies for assessment of HCV antibody and RNA levels have improved remarkably, as well as our understanding of how to best use these tests in patient management. This review aims to give an overview of the different serological and molecular methods employed in detecting HCV infection used nowadays. Additionally, the review gives an insight in the new molecular techniques that are being developed to improve the detection techniques particularly in High Risk Group population who are severely immunocompromised.
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37
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Hedskog C, Doehle B, Chodavarapu K, Gontcharova V, Crespo Garcia J, De Knegt R, Drenth JPH, McHutchison JG, Brainard D, Stamm LM, Miller MD, Svarovskaia E, Mo H. Characterization of hepatitis C virus intergenotypic recombinant strains and associated virological response to sofosbuvir/ribavirin. Hepatology 2015; 61:471-80. [PMID: 25099344 DOI: 10.1002/hep.27361] [Citation(s) in RCA: 72] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/24/2014] [Accepted: 08/01/2014] [Indexed: 12/31/2022]
Abstract
UNLABELLED To date, intergenotypic recombinant hepatitis C viruses (HCVs) and their treatment outcomes have not been well characterized. This study characterized 12 novel HCV recombinant strains and their response to sofosbuvir in combination with ribavirin (SOF/RBV) treatment. Across the phase II/III studies of SOF, HCV samples were genotyped using both the Siemens VERSANT HCV Genotype INNO-LiPA 2.0 Assay (Innogenetics, Ghent, Belgium) and nonstructural (NS)5B sequencing. Among these patient samples, genotype assignment discordance between the two methods was found in 0.5% of all cases (12 of 2,363), of which all were identified as genotype 2 by INNO-LiPA (12 of 487; 2.5%). HCV full-genome sequences were obtained for these 12 samples by a sequence-independent amplification method coupled with next-generation sequencing. HCV full-genome sequencing revealed that these viruses were recombinant HCV strains, with the 5' part corresponding to genotype 2 and the 3' part corresponding to genotype 1. The recombination breakpoint between genotypes 2 and 1 was consistently located within 80 amino acids of the NS2/NS3 junction. Interestingly, one of the recombinant viruses had a 34-amino-acid duplication at the location of the recombination breakpoint. Eleven of these twelve patients were treated with a regimen for genotype 2 HCV infection, but responded as if they had genotype 1 infection; 1 patient had received placebo. CONCLUSION Twelve new HCV intergenotypic recombinant genotype 2/1 viruses have been characterized. The antiviral response to a 12- to 16-week course of SOF/RBV treatment in these patients was more similar to responses among genotype 1 patients than genotype 2 patients, consistent with their genotype 1 NS5B gene.
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38
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Mukherjee R, Burns A, Rodden D, Chang F, Chaum M, Garcia N, Bollipalli N, Niemz A. Diagnosis and Management of Hepatitis C Virus Infection. ACTA ACUST UNITED AC 2015; 20:519-38. [PMID: 25609256 DOI: 10.1177/2211068214563794] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2014] [Indexed: 01/03/2023]
Abstract
The hepatitis C virus (HCV) infects more than 200 million people globally, with increasing incidence, especially in developing countries. HCV infection frequently progresses to chronic liver disease, creating a heavy economic burden on resource-poor countries and lowering patient quality of life. Effective HCV diagnosis, treatment selection, and treatment monitoring are important in stopping disease progression. Serological assays, which detect anti-HCV antibodies in the patient after seroconversion, are used for initial HCV diagnosis. Qualitative and quantitative molecular assays are used to confirm initial diagnosis, determine viral load, and genotype the dominant strain. Viral load and genotype information are used to guide appropriate treatment. Various other biomarker assays are performed to assess liver function and enable disease staging. Most of these diagnostic methods are mature and routinely used in high-resource countries with well-developed laboratory infrastructure. Few technologies, however, are available that address the needs of low-resource areas with high HCV prevalence, such as Africa and Southeast Asia.
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Affiliation(s)
- Ronita Mukherjee
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
| | - Andrew Burns
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
| | - Diane Rodden
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
| | - Frances Chang
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
| | - Manita Chaum
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
| | - Nancy Garcia
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
| | | | - Angelika Niemz
- Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
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39
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Quer J, Gregori J, Rodríguez-Frias F, Buti M, Madejon A, Perez-del-Pulgar S, Garcia-Cehic D, Casillas R, Blasi M, Homs M, Tabernero D, Alvarez-Tejado M, Muñoz JM, Cubero M, Caballero A, del Campo JA, Domingo E, Belmonte I, Nieto L, Lens S, Muñoz-de-Rueda P, Sanz-Cameno P, Sauleda S, Bes M, Gomez J, Briones C, Perales C, Sheldon J, Castells L, Viladomiu L, Salmeron J, Ruiz-Extremera A, Quiles-Pérez R, Moreno-Otero R, López-Rodríguez R, Allende H, Romero-Gómez M, Guardia J, Esteban R, Garcia-Samaniego J, Forns X, Esteban JI. High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods. J Clin Microbiol 2015; 53:219-226. [PMID: 25378574 PMCID: PMC4290919 DOI: 10.1128/jcm.02093-14] [Citation(s) in RCA: 66] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2014] [Accepted: 10/30/2014] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) is classified into seven major genotypes and 67 subtypes. Recent studies have shown that in HCV genotype 1-infected patients, response rates to regimens containing direct-acting antivirals (DAAs) are subtype dependent. Currently available genotyping methods have limited subtyping accuracy. We have evaluated the performance of a deep-sequencing-based HCV subtyping assay, developed for the 454/GS-Junior platform, in comparison with those of two commercial assays (Versant HCV genotype 2.0 and Abbott Real-time HCV Genotype II) and using direct NS5B sequencing as a gold standard (direct sequencing), in 114 clinical specimens previously tested by first-generation hybridization assay (82 genotype 1 and 32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 calling by population Sanger sequencing (69% 1b, 31% 1a) in 81 specimens and identified a mixed-subtype infection (1b/3a/1a) in one sample. Similarly, among the 32 previously indeterminate specimens, identical genotype and subtype results were obtained by direct and deep sequencing in all but four samples with dual infection. In contrast, both Versant HCV Genotype 2.0 and Abbott Real-time HCV Genotype II failed subtype 1 calling in 13 (16%) samples each and were unable to identify the HCV genotype and/or subtype in more than half of the non-genotype 1 samples. We concluded that deep sequencing is more efficient for HCV subtyping than currently available methods and allows qualitative identification of mixed infections and may be more helpful with respect to informing treatment strategies with new DAA-containing regimens across all HCV subtypes.
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Affiliation(s)
- Josep Quer
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Josep Gregori
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Roche Diagnostics SL, Barcelona, Spain
| | - Francisco Rodríguez-Frias
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - Maria Buti
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Antonio Madejon
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Liver Unit, Hospital La Paz-Carlos III, Madrid, Spain
| | - Sofia Perez-del-Pulgar
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Liver Unit, Hospital Clinic, IDIBAPS, Barcelona, Spain
| | - Damir Garcia-Cehic
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Rosario Casillas
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - Maria Blasi
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - Maria Homs
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - David Tabernero
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | | | | | - Maria Cubero
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Andrea Caballero
- Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - Jose Antonio del Campo
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital Universitario Virgen de Valme, Seville, Spain
| | - Esteban Domingo
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Centro de Biología Molecular Severo Ochoa-Universidad Autónoma de Madrid (CSIC-UAM), Campus de Cantoblanco, Madrid, Spain
| | - Irene Belmonte
- Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - Leonardo Nieto
- Biochemistry Unit, Virology Unit /Microbiology Department, HUVH, Barcelona, Spain
| | - Sabela Lens
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Liver Unit, Hospital Clinic, IDIBAPS, Barcelona, Spain
| | - Paloma Muñoz-de-Rueda
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital San Cecilio, Granada, Spain
| | - Paloma Sanz-Cameno
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital de la Princesa, Madrid, Spain
| | - Silvia Sauleda
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Banc de Sang i de Teixits, Institut Català de la Salut, Barcelona, Spain
| | - Marta Bes
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Banc de Sang i de Teixits, Institut Català de la Salut, Barcelona, Spain
| | - Jordi Gomez
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain CSIC, Instituto de Parasitología y Biomedicina López Neyra, Granada, Spain
| | - Carlos Briones
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Centro de Astrobiología (CSIC-INTA), Madrid, Spain
| | - Celia Perales
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Centro de Biología Molecular Severo Ochoa-Universidad Autónoma de Madrid (CSIC-UAM), Campus de Cantoblanco, Madrid, Spain
| | - Julie Sheldon
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Centro de Biología Molecular Severo Ochoa-Universidad Autónoma de Madrid (CSIC-UAM), Campus de Cantoblanco, Madrid, Spain
| | - Lluis Castells
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Lluis Viladomiu
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain
| | - Javier Salmeron
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital San Cecilio, Granada, Spain
| | - Angela Ruiz-Extremera
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital San Cecilio, Granada, Spain
| | - Rosa Quiles-Pérez
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital San Cecilio, Granada, Spain
| | - Ricardo Moreno-Otero
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital de la Princesa, Madrid, Spain
| | - Rosario López-Rodríguez
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital de la Princesa, Madrid, Spain
| | - Helena Allende
- Pathological Anatomy Department, VHIR-HUVH, Barcelona, Spain
| | - Manuel Romero-Gómez
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Hospital Universitario Virgen de Valme, Seville, Spain
| | - Jaume Guardia
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Rafael Esteban
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain
| | - Javier Garcia-Samaniego
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Liver Unit, Hospital La Paz-Carlos III, Madrid, Spain
| | - Xavier Forns
- Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Liver Unit, Hospital Clinic, IDIBAPS, Barcelona, Spain
| | - Juan Ignacio Esteban
- Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron (VHIR-HUVH), Barcelona, Spain Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) del Instituto de Salud Carlos III, Madrid, Spain Universitat Autònoma de Barcelona, Barcelona, Spain
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Hussein N, Zekri ARN, Abouelhoda M, Alam El-Din HM, Ghamry AA, Amer MA, Sherif GM, Bahnassy AA. New insight into HCV E1/E2 region of genotype 4a. Virol J 2014; 11:231. [PMID: 25547228 PMCID: PMC4304183 DOI: 10.1186/s12985-014-0231-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2014] [Accepted: 12/17/2014] [Indexed: 01/21/2023] Open
Abstract
Introduction Hepatitis C virus (HCV) genome contains two envelope proteins (E1 and E2) responsible for the virus entry into the cell. There is a substantial lack of sequences covering the full length of E1/E2 region for genotype 4. Our study aims at providing new sequences as well as characterizing the genetic divergence of the E1/E2 region of HCV 4a using our new sequences along with all publicly available datasets. Methods The genomic segments covering the whole E1/E2 region were isolated from Egyptian HCV patients and sequenced. The resulting 36 sequences 36 were analyzed using sequence analysis techniques to study variability within and among hosts in the same time point. Furthermore, previously published HCV E1/E2 sequence datasets for genotype 4a were retrieved and categorized according to the geographical location and date of isolation and were used for further analysis of variability among Egyptian over a period of 15 years, also compared with non-Egyptian sequences to figure out region-specific variability. Results Phylogenetic analysis of the new sequences has shown variability within the host and among different individuals in the same time point. Analysis of the 36 sequences along with the Egyptian sequences (254 sequences in E1 in the period from 1997 to 2010 and 8 E2 sequences in the period from 2006 to 2010) has shown temporal change over time. Analysis of the new HCV sequences with the non-Egyptian sequences (182 sequences in E1 and 155 sequences in the E2) has shown region specific variability. The molecular clock rate of E1 was estimated to be 5E-3 per site per year for Egyptian and 5.38E-3 for non-Egyptian. The clock rate of E2 was estimated to be 8.48E per site per year for Egyptian and 6.3E-3 for non-Egyptian. Conclusion The results of this study support the high rate of evolution of the Egyptian HCV genotype 4a. It has also revealed significant level of genetic variability among sequences from different regions in the world. Electronic supplementary material The online version of this article (doi:10.1186/s12985-014-0231-y) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Nehal Hussein
- Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Fom El-Khalig, Cairo, 11796, Egypt.
| | - Abdel-Rahman N Zekri
- Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Fom El-Khalig, Cairo, 11796, Egypt.
| | - Mohamed Abouelhoda
- Faculty of Engineering, Cairo University, Giza, Egypt. .,Center for Informatics Sciences, Nile University, Giza, Egypt.
| | - Hanaa M Alam El-Din
- Virology and Immunology Unit, Cancer Biology Department, National Cancer Institute, Cairo University, Fom El-Khalig, Cairo, 11796, Egypt.
| | | | - Mahmoud A Amer
- Faculty of Science, Zoology Department, Cairo University, Giza, Egypt.
| | - Ghada M Sherif
- Biostatistic & Epidemiology Department, National Cancer Institute, Cairo University, Cairo, Egypt.
| | - Abeer A Bahnassy
- Pathology Department, National Cancer Institute, Cairo University, Cairo, Egypt.
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Casanova YS, Boeira TDR, Sisti E, Celmer Á, Fonseca ASK, Ikuta N, Simon D, Lunge VR. A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping. Rev Soc Bras Med Trop 2014; 47:287-94. [PMID: 25075478 DOI: 10.1590/0037-8682-0040-2014] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2014] [Accepted: 06/16/2014] [Indexed: 12/11/2022] Open
Abstract
INTRODUCTION Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5' untranslated region (5'UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. METHODS Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. RESULTS The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. CONCLUSIONS A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping.
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Affiliation(s)
- Yara Silva Casanova
- Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil
| | | | - Elisa Sisti
- Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil
| | | | | | - Nilo Ikuta
- Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil
| | - Daniel Simon
- Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil
| | - Vagner Ricardo Lunge
- Laboratório de Diagnóstico Molecular, Programa de Pós-Graduação em Biologia Celular e Molecular Aplicada à Saúde, Universidade Luterana do Brasil, Canoas, RS, Brasil
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Cobb B, Heilek G, Vilchez RA. Molecular diagnostics in the management of chronic hepatitis C: key considerations in the era of new antiviral therapies. BMC Infect Dis 2014; 14 Suppl 5:S8. [PMID: 25236936 PMCID: PMC4160902 DOI: 10.1186/1471-2334-14-s5-s8] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Molecular tests that detect and/or quantify HCV RNA are important in the diagnosis and management of patients with chronic hepatitis C (CHC) undergoing anti-viral therapy. The primary goal of anti-HCV therapy is to achieve a sustained virologic response (SVR) defined as "undetectable" Hepatitis C Virus (HCV) RNA in the serum or plasma at 12 to 24 weeks following the end of treatment.
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Affiliation(s)
- Bryan Cobb
- Roche Molecular Systems Inc., Pleasanton, California, USA
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Gammal RS, Spooner LM, Abraham GM. Failed triple therapy in a treatment-experienced patient with genotype 6 hepatitis C infection. Am J Health Syst Pharm 2014; 71:204-8. [PMID: 24429013 DOI: 10.2146/ajhp130432] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
PURPOSE The first published report of the use of triple therapy in a patient with hepatitis C virus (HCV) genotype 6 infection-a treatment that was prescribed due to incorrect HCV genotyping and which ultimately failed-is presented. SUMMARY A 70-year-old male U.S. resident of Vietnamese descent requested treatment for chronic HCV infection acquired decades earlier. He reported experiencing hepatitis C treatment failures twice before-13 years prior (interferon alfa monotherapy for six months) and 7 years prior (standard dual therapy with pegylated interferon alfa-2b and ribavirin for nine months). Initial viral genotyping indicated infection with HCV genotypes 1a and 6c (a form of mixed HCV disease amenable to triple therapy), and treatment with pegylated interferon alfa-2a, ribavirin, and boceprevir was initiated. By week 8 of triple therapy, the patient's viral load had decreased from 15,700,000 (7.20 log) to 462,882 (5.67 log) IU/mL, but the viral load subsequently rebounded to baseline levels, and treatment was discontinued at week 16. When repeat HCV genotyping was performed, it was discovered that initial genotyping was incorrect and that the man's infection involved not mixed genotypes but only genotype 6; he was not an appropriate candidate for triple therapy. The case emphasizes the need for clinicians to be cognizant of potential HCV genotyping errors, particularly with regard to patients of Southeast Asian descent. CONCLUSION Three courses of interferon-based treatment, including triple therapy with boceprevir, failed to produce a sustained therapeutic response in a 70-year-old ethnic Vietnamese man with genotype 6 HCV infection.
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Affiliation(s)
- Roseann S Gammal
- Roseann S. Gammal, Pharm.D., is Postgraduate Year 1 Pharmacy Practice Resident, Department of Pharmacy, University of North Carolina Hospitals and Clinics, Chapel Hill. Linda M. Spooner, Pharm.D., BCPS, is Associate Professor of Pharmacy Practice, School of Pharmacy, MCPHS University, Worcester, MA. George M. Abraham, M.D., M.P.H., is Professor of Medicine, University of Massachusetts Medical School, Worcester, and Associate Chief of Medicine, Division of Infectious Disease and Geographic Medicine, Saint Vincent Hospital, Worcester
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Reassessment of genotype 1 hepatitis C virus subtype misclassification by LiPA 2.0: implications for direct-acting antiviral treatment. J Clin Microbiol 2014; 52:4027-9. [PMID: 25143567 DOI: 10.1128/jcm.02209-14] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The accuracy of LiPA 2.0 for hepatitis C virus 1 (HCV-1) subtype classification was analyzed. LiPA 2.0 genotype results from 101 HCV-1-infected patients were compared to genotype findings determined by direct core sequencing. Eleven (11%) samples were misclassified. Given the influence of the HCV-1-subtype in the anti-HCV therapy response, an alternative classification method is warranted.
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The need for a sequencing-based assay to supplement the Abbott m2000 RealTime HCV Genotype II assay: A 1 year analysis. J Clin Virol 2014; 60:301-4. [DOI: 10.1016/j.jcv.2014.04.005] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2014] [Revised: 03/29/2014] [Accepted: 04/06/2014] [Indexed: 02/05/2023]
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Back to the origin of HCV 2c subtype and spreading to the Calabria region (Southern Italy) over the last two centuries: a phylogenetic study. INFECTION GENETICS AND EVOLUTION 2014; 26:352-8. [PMID: 24973737 DOI: 10.1016/j.meegid.2014.06.006] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/24/2014] [Revised: 05/23/2014] [Accepted: 06/09/2014] [Indexed: 12/12/2022]
Abstract
Circulation of HCV genotype 2 has been described in European Countries where numerous subtypes and unclassified HCV 2 lineages have been reported. In Italy, subtype 1b is the most prevalent, followed by genotype 2. In the present study, phylogeny of HCV 2c was investigated. The phylogeny of HCV 2c isolated from 54 Italian patients in the Calabria region (Southern Italy) was investigated by analyzing a fragment of the NS5B gene. Patients came from 5 metropolitan areas and a small village (Sersale). These areas were geographically dispersed throughout the entire region. A Bayesian coalescent-based framework was used to estimate origin and spreading of HCV 2c in this region. Phylogenetic analysis showed that 28 Italian sequences were intermixed with foreign HCV 2c reference sequences and grouped into 3 major clades: A, B, and C. Nineteen inter-clade sequences were associated uniquely with surgery as risk factor for HCV acquisition. By contrast, a sub-cluster within clade B was associated with blood transfusion. Moreover, sequences from Sersale village grouped in the Italian sub-cluster and were intermixed with 10 sequences from metropolitan areas. The three isolates with the longest branch came from Sersale and belonged to patients who had glass syringes as risk factor. HCV 2c isolates from the Calabria region shared a common ancestor whose origin was traced back to 1889. Our results suggest that, after its introduction - possibly as a result of population movements between Italy and African Countries during Italian colonialism - HCV 2c spread through multiple risk factors, not including intravenous drug use. So, transmission chains followed a pathway different from other European Countries. Although HCV incidence is decreasing, these ways are still ongoing, possibly justifying stability in the relative prevalence of HCV 2c.
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Clinical Aspects of Hepatitis C Virus Infection. Antiviral Res 2014. [DOI: 10.1128/9781555815493.ch14] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Saludes V, González V, Planas R, Matas L, Ausina V, Martró E. Tools for the diagnosis of hepatitis C virus infection and hepatic fibrosis staging. World J Gastroenterol 2014; 20:3431-3442. [PMID: 24707126 PMCID: PMC3974510 DOI: 10.3748/wjg.v20.i13.3431] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Revised: 12/05/2013] [Accepted: 03/06/2014] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infection represents a major public health issue. Hepatitis C can be cured by therapy, but many infected individuals are unaware of their status. Effective HCV screening, fast diagnosis and characterization, and hepatic fibrosis staging are highly relevant for controlling transmission, treating infected patients and, consequently, avoiding end-stage liver disease. Exposure to HCV can be determined with high sensitivity and specificity with currently available third generation serology assays. Additionally, the use of point-of-care tests can increase HCV screening opportunities. However, active HCV infection must be confirmed by direct diagnosis methods. Additionally, HCV genotyping is required prior to starting any treatment. Increasingly, high-volume clinical laboratories use different types of automated platforms, which have simplified sample processing, reduced hands-on-time, minimized contamination risks and human error and ensured full traceability of results. Significant advances have also been made in the field of fibrosis stage assessment with the development of non-invasive methods, such as imaging techniques and serum-based tests. However, no single test is currently available that is able to completely replace liver biopsy. This review focuses on approved commercial tools used to diagnose HCV infection and the recommended hepatic fibrosis staging tests.
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Thong VD, Akkarathamrongsin S, Poovorawan K, Tangkijvanich P, Poovorawan Y. Hepatitis C virus genotype 6: virology, epidemiology, genetic variation and clinical implication. World J Gastroenterol 2014; 20:2927-2940. [PMID: 24659883 PMCID: PMC3961978 DOI: 10.3748/wjg.v20.i11.2927] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2013] [Revised: 01/06/2014] [Accepted: 01/19/2014] [Indexed: 02/07/2023] Open
Abstract
Hepatitis C virus (HCV) is a serious public health problem affecting 170 million carriers worldwide. It is a leading cause of chronic hepatitis, cirrhosis, and liver cancer and is the primary cause for liver transplantation worldwide. HCV genotype 6 (HCV-6) is restricted to South China, South-East Asia, and it is also occasionally found in migrant patients from endemic countries. HCV-6 has considerable genetic diversity with 23 subtypes (a to w). Although direct sequencing followed by phylogenetic analysis is the gold standard for HCV-6 genotyping and subtyping, there are also now rapid genotyping tests available such as the reverse hybridization line probe assay (INNO-LiPA II; Innogenetics, Zwijnaarde, Belgium). HCV-6 patients present with similar clinical manifestations as patients infected with other genotypes. Based on current evidence, the optimal treatment duration of HCV-6 with pegylated interferon/ribavirin should be 48 wk, although a shortened treatment duration of 24 wk could be sufficient in patients with low pretreatment viral load who achieve rapid virological response. In addition, the development of direct-acting antiviral agents is ongoing, and they give high response rate when combined with standard therapy. Herein, we review the epidemiology, classification, diagnosis and treatment as it pertain to HCV-6.
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Mallory MA, Lucic DX, Sears MT, Cloherty GA, Hillyard DR. Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing. J Clin Virol 2014; 60:22-6. [PMID: 24656214 DOI: 10.1016/j.jcv.2014.02.006] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2013] [Revised: 02/06/2014] [Accepted: 02/17/2014] [Indexed: 02/02/2023]
Abstract
BACKGROUND HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. OBJECTIVE To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. STUDY DESIGN The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. RESULTS Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. CONCLUSIONS The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method.
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Affiliation(s)
- Melanie A Mallory
- ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
| | - Danijela X Lucic
- Abbott Molecular Inc., 1350 E. Touhy Avenue, Des Plaines, IL 60018, USA.
| | - Mitchell T Sears
- ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
| | - Gavin A Cloherty
- Abbott Molecular Inc., 1350 E. Touhy Avenue, Des Plaines, IL 60018, USA.
| | - David R Hillyard
- ARUP Institute for Clinical and Experimental Pathology, ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108, USA; University of Utah Department of Pathology, 15 North Medical Drive East, Salt Lake City, UT 84112, USA.
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