title: Multitypic hepatitis C virus infection identified by real-time nucleotide sequencing of minority genotypes.
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.
authors.affiliation: Centre for Infections, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT, United Kingdom. andrew.buckton@hpa.org.uk
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Buckton
authors.forename: Siew-Lin
authors.initials: SL
authors.lastname: Ngui
authors.forename: Catherine
authors.initials: C
authors.lastname: Arnold
authors.forename: Katie
authors.initials: K
authors.lastname: Boast
authors.forename: Joanne
authors.initials: J
authors.lastname: Kovacs
authors.forename: Paul E
authors.initials: PE
authors.lastname: Klapper
authors.forename: Bharat
authors.initials: B
authors.lastname: Patel
authors.forename: Imad
authors.initials: I
authors.lastname: Ibrahim
authors.forename: Savita
authors.initials: S
authors.lastname: Rangarajan
authors.forename: Mary E
authors.initials: ME
authors.lastname: Ramsay
authors.forename: Chong-Gee
authors.initials: CG
authors.lastname: Teo
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000328:Adult; D000368:Aged; D001483:Base Sequence; D001769:Blood / Q000821:virology; D025861:Blood Coagulation Disorders, Inherited / Q000150:complications; D003001:Cloning, Molecular / Q000379:methods*; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008875:Middle Aged; D012367:RNA, Viral / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D015819:Substance Abuse, Intravenous / Q000150:complications
chemical_list: D020121:5' Untranslated Regions; D012367:RNA, Viral