Evaluation of a reverse hybridization assay for genotyping of hepatitis C virus.
OBJECTIVE
Several strains of the hepatitis C virus exist; distinct genotypes and subtypes can be identified by sequence comparison of the viral genomes. Recent evidence that the genotype/subtype of hepatitis C virus may influence the clinical course of chronic hepatitis C and the response to interferon-alpha therapy for this disease suggests that methods to identify the genotype may become clinically useful. In the present study we evaluated a recently introduced reverse hybridization assay.


METHODS
HCV-RNA was isolated from serum samples from 61 consecutive patients attending our out-patient clinic and subsequently sequenced in the 5'-noncoding and the nonstructural-5 region by the dideoxynucleotide chain termination method. HCV-genotyping was performed by phylogenetic analysis of nonstructural-5 sequences. The amplification product for the reverse hybridization assay was obtained by "nested" polymerase chain reaction using biotinylated primers corresponding to the 5'-noncoding region. The assay is based on hybridization of the resulting polymerase chain reaction product with oligonucleotide probes immobilized as parallel lines on membrane strips.


RESULTS
According to the phylogenetic analysis of the nonstructural-5 region the prevalence of hepatitis C virus subtypes was as follows: 1a 18%, 1b 51%, 2a 3%, 2b 3%, 2c 7% and 3a 18%. The reverse hybridization assay correctly identified each hepatitis C virus genotype (1, 2, and 3). However, differentiation of hepatitis C virus subtypes was insufficient. 1/11 HCV-1a isolates was incorrectly classified by the reverse hybridization assay as HCV-1b and vice versa 3/31 HCV-1b isolates as HCV-1a. Classification of hepatitis C virus subtypes 2a, 2b and 3a was correct, but 4/4 HCV-2c isolates were misinterpreted by the assay as HCV-2a.


CONCLUSIONS
The reverse hybridization assay can differentiate between hepatitis C virus genotypes 1, 2, and 3, but is not completely reliable for hepatitis C virus subtyping.
Zeuzem
S
S
Medical Department II, University Hospital, Frankfurt, Germany.
Ruster
B
B
Lee
J H
JH
Stripf
T
T
Roth
W K
WK
D001483:Base Sequence; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction