title: Ultracentrifugation of serum samples allows detection of hepatitis C virus RNA in patients with occult hepatitis C.
abstract: Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.
authors.affiliation: Fundación Estudio Hepatitis Virales, Madrid, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Bartolomé
authors.forename: Juan Manuel
authors.initials: JM
authors.lastname: López-Alcorocho
authors.forename: Inmaculada
authors.initials: I
authors.lastname: Castillo
authors.forename: Elena
authors.initials: E
authors.lastname: Rodríguez-Iñigo
authors.forename: Juan Antonio
authors.initials: JA
authors.lastname: Quiroga
authors.forename: Ricardo
authors.initials: R
authors.lastname: Palacios
authors.forename: Vicente
authors.initials: V
authors.lastname: Carreño
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005260:Female; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D017403:In Situ Hybridization; D008297:Male; D010802:Phylogeny; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014461:Ultracentrifugation
chemical_list: D017931:DNA Primers; D012367:RNA, Viral