Hepatitis C virus heteroduplex tracking assay for genotype determination reveals diverging genotype 2 isolates in Italian hemodialysis patients.
A heteroduplex tracking assay (HTA) was developed for genetic analyses of the hepatitis C virus (HCV) using single-stranded probes from the core (C)/E1 region. Nucleotide sequencing of reverse transcriptase (RT)-PCR products from 15 Italian dialysis patients confirmed the specificity and accuracy of the HTA genotyping method, which identified 5 of 15 (33.3%) 1b, 7 of 15 (46.7%) 3a, and 3 of 15 (20%) type 2 infections. The genotypes of an additional 12 HCV antibody-positive blood donors from different geographical locations were also in agreement with the genotypes determined by the Inno-LiPA HCV II kit (Innogenetics) and/or restriction fragment length polymorphism (RFLP). Isolates which had between 35 to 40% nucleotide divergence from control subtype 1a, 1b, 2a, 2b, or 3a standards could be typed. Surprisingly, HTA detected one 1b-2 coinfection which was missed by DNA sequencing. Three samples that were designated non-2a or 2b type 2 by HTA were found to be type 2a by both RFLP and direct nucleotide sequencing of the 5' untranslated region. The genetic distance between patient type 2 and control 2a, 2b, and 2c isolates indicated that a new subtype was present in the population being studied. Serotyping (RIBA serotyping strip immunoblot assay kit) of 23 dialysis patients showed that the genotype could be determined in 6 of 8 (75%) C/E1 RT-PCR-negative and 15 of 23 (65.2%) RT-PCR-positive samples, indicating that the two tests complement each other.
Calvo
P L
PL
Blood Bank, Molinette Hospital, Turin, Italy.
Kansopon
J
J
Sra
K
K
Quan
S
S
DiNello
R
R
Guaschino
R
R
Calabrese
G
G
Danielle
F
F
Brunetto
M R
MR
Bonino
F
F
Massaro
A L
AL
Polito
A
A
Houghton
M
M
Weiner
A J
AJ
D001782:Blood Donors; D004279:DNA, Viral / Q000737:chemistry; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics; D006801:Humans; D012150:Polymorphism, Restriction Fragment Length; D006435:Renal Dialysis; D012680:Sensitivity and Specificity; D012703:Serotyping
D004279:DNA, Viral