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.
Buckton
Andrew J
AJ
Centre for Infections, Health Protection Agency, 61 Colindale Avenue, Colindale, London NW9 5HT, United Kingdom. andrew.buckton@hpa.org.uk
Ngui
Siew-Lin
SL
Arnold
Catherine
C
Boast
Katie
K
Kovacs
Joanne
J
Klapper
Paul E
PE
Patel
Bharat
B
Ibrahim
Imad
I
Rangarajan
Savita
S
Ramsay
Mary E
ME
Teo
Chong-Gee
CG
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
D020121:5' Untranslated Regions; D012367:RNA, Viral