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 originwho 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 ؋ 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. The viral genome of hepatitis C virus (HCV) is a 9.6-kb linear single-stranded RNA molecule of positive polarity that replicates via an HCV RNA strand of negative polarity (18).Chronic HCV infection is a progressive disease that may lead to liver cirrhosis and hepatocellular carcinoma (6, 21). The hallmark of chronic hepatitis C is the presence of anti-HCV and HCV RNA in serum for more than 6 months after acute infection. Recently, a new form of chronic HCV infection called "occult HCV infection," characterized by the presence of genomic HCV RNA in liver in the absence of anti-HCV and serum HCV RNA, in patients with abnormal liver function tests of unknown etiology was described (3). In 84% of patients with occult HCV infection, the negative-polarity (antigenomic) HCV RNA strand was also detected in liver, indicating that the virus was replicating. Furthermore, 70% of these patients had HCV RNA in peripheral blood mononuclear cells (PBMCs) (3), and viral replication in these cells was also reported (4).Since HCV replicates in the livers of patients with occult HCV infection, the reason why HCV RNA is not detected in serum is unknown. One possible explanation is that the number of circulating viral particles is too low to be detected even using the most sensitive reverse transcription (RT)-PCR techniques.In the present study, by using ultracentrifugation and a sensitive real-time PCR technique, we have demonstrated that sera from patients with occult HCV infection contain low amounts of viral particles and that the physical characteristics of these virions are similar to those found in patients with classical chronic HCV infection. MATERIALS AND METHODS Patients.One hundred six patients (74 males) with occult HCV infection were analyzed in this study. Patients had abnormal liver function tests, were anti-HCV negative (INNOTEST HCV Ab IV; Innogenetics, Ghent, Belgium), and did not have serum HCV RNA levels tested by real-time RT-PCR (see below). Other causes of liver disease...

Occult Hepatitis C Virus Infection among Hemodialysis Patients

“…Primers and conditions of this strand-specific realtime PCR have been reported. 13 The fluorescence resonance energy transfer probes used were described by Bullock et al 14 These allow discrimination between HCV genotypes 1, 2, 3, and 4 by melting-curve analysis. Quantification of both HCV-RNA strands was performed using two standard curves constructed with 10-fold dilutions of synthetic genomic and antigenomic HCV-RNA (from 3.2 ϫ 10 8 to 0.32 copies).…”

“…This assay was capable of detecting 3.2 molecules of the correct strand while unspecifically detecting 10 7 to 10 8 copies of the incorrect strand. 13 For ensuring the specificity of the results, HCV-RNA detection was performed blindly on different days by two different operators. In addition to PBMC from the 10 healthy anti-HCV negative patients, total RNA isolated from HepG2 cells and blanks were used as negative controls.…”

When to Search for Occult Infection After Eradication of Hepatitis C?

“…It has been proposed that the number of circulating viral particles is too low to be detected by conventional molecular techniques. 5 Although several authors have described this new form of infection with emerging implications in different clinical scenarios, 6 few data are available in the setting of liver transplantation.…”

Histological changes in hepatitis C virus antibody-positive, hepatitis C virus RNA-negative subjects suggest persistent virus infection

“…All these specimens may well represent true HCV RNA-positive samples in which viral breakthrough was serendipitously identified; however, the supporting evidence is lacking. Additionally, the authors should have tested liver biopsy material to support the supposition made in the article title.The existence of seropositive and seronegative aviremic occult HCV has been proposed by Carreno et al [3][4][5] These patients often have periodic elevations of alanine aminotransferase. Hoare et al 1 reported that 15.2% of the study group had alanine aminotransferase elevations; perhaps these latter individuals would have been a more appropriate study group.…”

“…The existence of seropositive and seronegative aviremic occult HCV has been proposed by Carreno et al [3][4][5] These patients often have periodic elevations of alanine aminotransferase. Hoare et al 1 reported that 15.2% of the study group had alanine aminotransferase elevations; perhaps these latter individuals would have been a more appropriate study group.…”