Comparison of hepatitis C virus subtyping by ns5b sequencing with 5'utr based methods.
OBJECTIVE
We compared Hepatitis C virus (HCV) genotyping by direct sequencing of the non-structural 5b region (NS5b) and a commercial PCR/hybridization method based on the conserved 5´-untranslated region (5'UTR).


METHODS
One hundred twenty HCV containing plasma samples were analyzed by NS5b sequencing with focus on samples with undetermined results or 1b subtype identification in the used combination of Cobas® AmpliPrep/Cobas® TaqMan96® PCR and subsequent Versant® HCV Genotype 2.0 Assay (LiPA).


RESULTS
There was 100% concordance between the two methods for genotyping but only 83% for subtyping. Seventeen samples were designated 1b by hybridization but subtype 1a by NS5b sequencing. This is a general 5'UTR problem as the discordant results were additionally confirmed by 5'UTR sequencing. Thus our routine combination not only misclassified 38.6% of subtype 1a isolates as 1b but in contrast to NS5b sequencing was unable to discriminate between subtypes 2a/c, or 4a/c/d and also failed on a newly described subtype (10a/3k). [corrected].


CONCLUSIONS
[corrected] The applied 5'UTR methods allow the rapid determination of HCV genotypes but failed to correctly identify the subtype in many samples. This has implications for epidemiological studies or forensic evaluation of chains of infection and NS5b sequencing therefore is our method of choice under those circumstances.
Laboratory for Microbiology and Virology, Azienda Sanitaria dell'Alto Adige, Bolzano, Italy. elisabetta.pagani@asbz.it
E
E
Pagani
H P
HP
Huemer
V
V
Pasquetto
C
C
Cemin
L
L
Molon
P
P
Rossi
C
C
Larcher
D020121:5' Untranslated Regions / Q000235:genetics*; D055028:Comparative Genomic Hybridization / Q000379:methods; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods
D020121:5' Untranslated Regions