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Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes

Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes
Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes

The prevalence of concurrent multitypic hepatitis C virus (HCV) infection is uncertain. A sensitive and specific approach to identifying minority HCV genotypes in blood is presented. Following serum extraction and reverse transcription PCR to amplify cDNA originating from the viral 5' noncoding region, the amplified product mixture was treated with genotype-specific restriction endonuclease to digest the dominant genotype. Residual amplicons were subjected to PCR cloning and then to real-time DNA sequencing using a Pyrosequencer to identify the remaining genotypes. Dilution experiments showed that minority genotypes may be detected when they represent 1:10,000 of the total population and in serum specimens with viral loads as low as 1,000 IU/ml. Of 37 patients with bleeding disorders and 44 injecting drug users, infection by more than one HCV genotype was found in 7 (19%) and 4 (9%) patients, respectively. The low rate of detection in people at high risk of repeated HCV infection suggests that multitypic HCV carriage is uncommon.

5' Untranslated Regions/genetics, Adult, Aged, Base Sequence, Blood/virology, Blood Coagulation Disorders, Inherited/complications, Cloning, Molecular/methods, Genotype, Hepacivirus/classification, Hepatitis C/virology, Humans, Middle Aged, RNA, Viral/genetics, Sensitivity and Specificity, Sequence Analysis, DNA/methods, Substance Abuse, Intravenous/complications
0095-1137
2779-2784
Buckton, Andrew J
d4a34164-8f2d-481f-861d-823cf5430207
Ngui, Siew-Lin
53f4119a-cee0-4fa2-9bf7-b5114450e529
Arnold, Catherine
96290dec-b659-4bb6-85a9-a65fe4341595
Boast, Katie
9a7ef8c3-3e52-4069-8d03-943ce8e47e36
Kovacs, Joanne
13eb9894-c19d-433b-9bab-2adb86e631f7
Klapper, Paul E
32fe9942-41f5-42dd-afac-e6db00b42b0f
Patel, Bharat
e95de217-e2ee-45a4-8765-d32a0195668e
Ibrahim, Imad
5216fa04-f1e1-458f-a42d-53d31718e752
Rangarajan, Savita
9a5e4c7e-55ba-4a3a-b5f6-f1e269d927c3
Ramsay, Mary E
8320d895-e9d3-4abc-862e-fe923a4ac69d
Teo, Chong-Gee
de675290-36d2-4680-9924-d8a2e545e47d
Buckton, Andrew J
d4a34164-8f2d-481f-861d-823cf5430207
Ngui, Siew-Lin
53f4119a-cee0-4fa2-9bf7-b5114450e529
Arnold, Catherine
96290dec-b659-4bb6-85a9-a65fe4341595
Boast, Katie
9a7ef8c3-3e52-4069-8d03-943ce8e47e36
Kovacs, Joanne
13eb9894-c19d-433b-9bab-2adb86e631f7
Klapper, Paul E
32fe9942-41f5-42dd-afac-e6db00b42b0f
Patel, Bharat
e95de217-e2ee-45a4-8765-d32a0195668e
Ibrahim, Imad
5216fa04-f1e1-458f-a42d-53d31718e752
Rangarajan, Savita
9a5e4c7e-55ba-4a3a-b5f6-f1e269d927c3
Ramsay, Mary E
8320d895-e9d3-4abc-862e-fe923a4ac69d
Teo, Chong-Gee
de675290-36d2-4680-9924-d8a2e545e47d

Buckton, Andrew J, Ngui, Siew-Lin, Arnold, Catherine, Boast, Katie, Kovacs, Joanne, Klapper, Paul E, Patel, Bharat, Ibrahim, Imad, Rangarajan, Savita, Ramsay, Mary E and Teo, Chong-Gee (2006) Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes. Journal of Clinical Microbiology, 44 (8), 2779-2784. (doi:10.1128/JCM.01638-05).

Record type: Article

Abstract

The prevalence of concurrent multitypic hepatitis C virus (HCV) infection is uncertain. A sensitive and specific approach to identifying minority HCV genotypes in blood is presented. Following serum extraction and reverse transcription PCR to amplify cDNA originating from the viral 5' noncoding region, the amplified product mixture was treated with genotype-specific restriction endonuclease to digest the dominant genotype. Residual amplicons were subjected to PCR cloning and then to real-time DNA sequencing using a Pyrosequencer to identify the remaining genotypes. Dilution experiments showed that minority genotypes may be detected when they represent 1:10,000 of the total population and in serum specimens with viral loads as low as 1,000 IU/ml. Of 37 patients with bleeding disorders and 44 injecting drug users, infection by more than one HCV genotype was found in 7 (19%) and 4 (9%) patients, respectively. The low rate of detection in people at high risk of repeated HCV infection suggests that multitypic HCV carriage is uncommon.

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More information

Published date: August 2006
Keywords: 5' Untranslated Regions/genetics, Adult, Aged, Base Sequence, Blood/virology, Blood Coagulation Disorders, Inherited/complications, Cloning, Molecular/methods, Genotype, Hepacivirus/classification, Hepatitis C/virology, Humans, Middle Aged, RNA, Viral/genetics, Sensitivity and Specificity, Sequence Analysis, DNA/methods, Substance Abuse, Intravenous/complications

Identifiers

Local EPrints ID: 443326
URI: http://eprints.soton.ac.uk/id/eprint/443326
ISSN: 0095-1137
PURE UUID: 181c781c-bd4c-42c1-8799-fb7f5a1131c9
ORCID for Savita Rangarajan: ORCID iD orcid.org/0000-0001-7367-133X

Catalogue record

Date deposited: 20 Aug 2020 16:33
Last modified: 17 Mar 2024 04:02

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Contributors

Author: Andrew J Buckton
Author: Siew-Lin Ngui
Author: Catherine Arnold
Author: Katie Boast
Author: Joanne Kovacs
Author: Paul E Klapper
Author: Bharat Patel
Author: Imad Ibrahim
Author: Mary E Ramsay
Author: Chong-Gee Teo

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