Javier Bartolomé

Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown.

BACKGROUND There are patients in whom the etiology of long-standing abnormal results of liver-function tests is unknown (ALF-EU) after exclusion of all known causes of liver diseases. We analyzed the presence of hepatitis C virus (HCV) RNA in liver-biopsy specimens from 100 patients who were negative for anti-HCV antibodies and for serum HCV RNA and who had ALF-EU. METHODS HCV RNA status was tested by reverse-transcription polymerase chain reaction (RT-PCR) and by in situ hybridization, in liver and peripheral-blood mononuclear cells (PBMCs). RESULTS HCV RNA was detected in liver-biopsy specimens from 57 of 100 patients negative for anti-HCV antibodies and for serum HCV RNA (i.e., who had occult HCV infection). HCV RNA of negative polarity was found in the liver of 48 (84.2%) of these 57 patients with occult HCV infection. Nucleotide-sequence analysis confirmed the specificity of detection of HCV RNA and that patients were infected with the HCV 1b genotype. Of these 57 patients with intrahepatic HCV RNA, 40 (70%) had viral RNA in their PBMCs. With regard to liver histology, patients with occult HCV infection were more likely to have necroinflammatory activity (P=.017) and fibrosis (P=.022) than were patients without intrahepatic HCV RNA. CONCLUSIONS Patients with ALF-EU may have intrahepatic HCV RNA in the absence of anti-HCV antibodies and of serum HCV RNA.

In Situ Detection of Hepatitis C Virus RNA in Salivary Glands

Chronic hepatitis C virus (HCV) infection has been associated with several extrahepatic manifestations, among these, to diseases with oral manifestations such as Sjögrens syndrome or sialadenitis. HCV-RNA has been detected in saliva and in salivary glands from patients with sialadenitis by polymerase chain reaction. However, morphological evidence of HCV replication in salivary gland cells is needed to support a role for HCV in causing sialadenitis or Sjögrens syndrome. We have used in situ hybridization and immunohistochemistry to analyze the presence of HCV-RNA of sense and antisense polarity and HCV core antigen, respectively, in salivary gland biopsies from 19 patients with chronic sialadenitis or Sjögrens syndrome (eight anti-HCV-positive; 11 anti-HCV-negative). HCV-RNA of both positive and negative polarity as well as HCV core antigen were detected in the epithelial cells of the salivary gland biopsies from all of the anti-HCV-positive patients but in none of the anti-HCV-negative cases. The percentage of HCV-infected cells ranged from 25 to 48.8% in the patients studied. In conclusion, we have shown that HCV infects and replicates in the epithelial cells from salivary glands of patients with Sjögrens syndrome or chronic sialadenitis. However, its implication in the pathogenesis of these diseases deserves future research.

Hepatitis C virus replicates in peripheral blood mononuclear cells of patients with occult hepatitis C virus infection

Background: Occult hepatitis C virus (HCV) infection is characterised by the presence of HCV-RNA in the liver in the absence of anti-HCV, and serum viral RNA. Up to 70% of these patients also have HCV-RNA in peripheral blood mononuclear cells (PBMC) but it is not known if HCV is replicating in these cells. Aim: We studied possible HCV replication in PBMC of 18 patients with an occult HCV infection who were selected on the basis of HCV-RNA positivity in PBMC. Methods: Detection of HCV-RNA positive and negative strands in PBMC was done by strand specific reverse transcriptase-polymerase chain reaction (RT-PCR) and by in situ hybridisation. Results: The presence of HCV-RNA positive strand in PBMC was confirmed in all patients by strand specific RT-PCR and by in situ hybridisation. Mean percentage of PBMC which had the HCV-RNA positive strand was 3.3% (95% confidence interval (CI) 2.1–4.4) The HCV-RNA negative strand was found in the PBMC of 11/18 (61%) patients by strand specific RT-PCR and confirmed by in situ hybridisation, and the percentage of PBMC harbouring the HCV-RNA negative strand was 3.1% (95% CI 0.8–5.5). There was a significant correlation (p = 0.001, r = 0.84) between the percentage of PBMC with the HCV-RNA positive strand and that of PBMC with the HCV-RNA negative strand. Conclusion: HCV replicates in the PBMC of patients with occult HCV infection and thus, although these patients do not have serum HCV-RNA, they could be potentially infectious.

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 × gav, 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 × 104 [range, 7.9 × 102 to 1.0 × 106] versus 2.3 × 104 [range, 4.0 × 102 to 2.2 × 105]) 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.

Long-term follow-up of hepatitis B chronic carriers who responded to interferon therapy

We studied the long-term outcome of patients with chronic hepatitis B virus (HBV) infection who responded to interferon (IFN) therapy. Between 1983 and 1988, 120 patients were included in 5 different protocols; 94 patients were treated with IFN and 26 were controls. Loss of serum HBV-DNA was considered a partial response and occurred in 34 of the treated patients and in 10 of the controls. Only the partial-response patients were followed up for 14-64 months (mean 46 months). HBeAg disappeared in 32/34 of the partial-response treated patients and in 9/10 of the controls. During the follow-up period, 6/34 (18%) treated patients and 1/10 controls suffered a reactivation of the disease with reappearance of HBV-DNA. Only 8/34 (23%) treated patients and 1/10 of the controls lost HBsAg; no statistical differences were observed in baseline characteristics between HBsAg-negative patients and patients who remained HBsAg-positive. Of eight HBsAg-negative treated patients, four were serum HBV-DNA-negative upon polymerase chain reaction and thus formed the HBsAg-negative control cases. Although the frequency of HBsAg loss in treated patients is relatively low, the improvement in liver disease obtained from IFN therapy is sustained over a long period.

Occult Hepatitis C Virus Infection among Hemodialysis Patients

Occult hepatitis C virus (HCV) infection (i.e., detectable HCV-RNA in the liver or peripheral blood mononuclear cells) in the absence of both serum HCV-RNA and anti-HCV antibodies has not been investigated in hemodialysis patients. In this study, real-time PCR and in situ hybridization was used to test for the presence of genomic and antigenomic HCV-RNA in peripheral blood mononuclear cells of 109 hemodialysis patients with abnormal levels of liver enzymes. Occult HCV infection, determined by the presence of genomic HCV-RNA, was found in 45% of the patients; 53% of these patients had ongoing HCV replication, indicated by the presence of antigenomic HCV-RNA. Patients with occult HCV infection had spent a significantly longer time on hemodialysis and had significantly higher mean alanine aminotransferase levels during the 6 mo before study entry. Logistic regression analysis revealed that mortality was associated with age >60 yr (odds ratio 3.30; 95% confidence interval 1.05 to 10.33) and the presence of occult HCV infection (odds ratio 3.84; 95% confidence interval 1.29 to 11.43). In conclusion, the prevalence of occult HCV infection is high among hemodialysis patients with persistently abnormal values of liver enzymes of unknown cause. The clinical significance of occult HCV infection in these patients requires further study.

Detection of hepatitis C virus (HCV) RNA in the liver of healthy, anti-HCV antibody-positive, serum HCV RNA-negative patients with normal alanine aminotransferase levels

BACKGROUND It is unknown whether hepatitis C virus (HCV) is present in the liver of anti-HCV antibody-positive patients with persistently normal alanine aminotransferase (ALT) levels and undetectable serum HCV RNA levels. METHODS We determined the presence of genomic and antigenomic HCV RNA strands in liver biopsy specimens and peripheral blood mononuclear cell (PBMC) samples obtained from 12 anti-HCV antibody-positive patients who had normal ALT levels and who had been serum HCV RNA negative for at least 12 months, according to the results of quantitative, strand-specific, real-time reverse-transcription-polymerase chain reaction and, also, in situ hybridization of liver cells. Intrahepatic HCV RNA was cloned and sequenced. RESULTS All patients remained anti-HCV antibody positive and serum HCV RNA negative, and all had normal ALT values during follow-up (mean duration +/- SD, 29.2 +/- 19.8 months). Genomic HCV RNA was detected in liver biopsy specimens obtained from 10 (83%) of 12 patients, and the antigenomic strand was detected in 10 (100%) of 10 liver biopsy specimens in which genomic HCV RNA was detected. Results were confirmed by in situ hybridization. Intrahepatic HCV was of genotype 1b, and HCV sequencing demonstrated no cross-contamination among samples. Genomic HCV RNA was found in 6 (50%) of 12 PBMC samples, and antigenomic HCV RNA was also detected in 5 (83%) of these 6 PBMC samples. CONCLUSION HCV may persist and replicate in the liver and PBMCs of healthy, anti-HCV antibody-positive, serum HCV RNA-negative patients who have persistently normal ALT levels. These patients should be followed up, because they have an ongoing viral infection.

Occult hepatitis B virus and hepatitis C virus infections

Occult HBV infection is a well‐recognised clinical entity characterised by the detection of HBV‐DNA in serum and/or in liver in the absence of detectable hepatitis B surface antigen (HBsAg). Occult HBV infection has been described not only in patients who have resolved an acute or chronic HBV infection but also in patients without any serological markers of a past HBV infection. Occult HBV infection in patients with chronic HCV infection may induce more severe liver disease and lower response rate to interferon treatment. The existence of occult HCV infections has been also reported more recently. Occult HCV infection is characterised by the presence of HCV‐RNA in liver and peripheral blood mononuclear cells in the absence of detectable serum HCV‐RNA. Occult HCV infection may occur under two different clinical situations: in hepatitis C antibody‐(anti‐HCV) negative and serum HCV‐RNA‐negative patients with abnormal liver function tests and in anti‐HCV‐positive patients who have no detectable serum HCV‐RNA and who have normal liver enzymes. The clinical relevance of occult HCV infections is still under investigation. Copyright

A Controlled Trial of Recombinant Interferon-Alpha in Caucasian Children with Chronic Hepatitis B

Twenty-four children with chronic active hepatitis due to hepatitis B virus (HBV) infection, who were positive for HBeAg and had increased levels of transaminases, were included in a controlled study of treatment using recombinant interferon-alpha (rIFN-alpha), 10 MU/m2 body surface, intramuscularly, 3 times a week over a period of 3 months. During therapy, a significant decrease in HBV-DNAp was observed in the 12 patients treated. By the end of therapy, the HBV-DNA had disappeared in 3 children, the same occurring in 1 child (33% overall) during the course of the 4th month. By this time, all the controls remained with HBV replication markers (p less than 0.05). The 4 treated patients who responded became HBeAg-negative, developing anti-HBe during the first 12 months after therapy. In the control group, the HBV-DNA disappeared in 3 children in the 7th month of follow-up. All of the children remained HBsAg-positive. The therapy with rIFN-alpha was well tolerated, secondary effects consisting of a flu-like syndrome and a slight decrease in leukocytes and platelets. At the second biopsy, 15 months after the beginning of therapy, a significant decrease in Knodells index of histological activity was observed in the responders. In the light of these results and since treated children lost viral replication markers in a shorter period of time than the controls, who seroconverted spontaneously, we consider that rIFN-alpha may be useful in the treatment of chronic hepatitis B in childhood.

Histological damage in chronic hepatitis C is not related to the extent of infection in the liver.

It has not been completely elucidated whether the liver injury induced by the hepatitis C virus (HCV) is due to direct cytopathic damage or to an immune-mediated response against HCV-infected hepatocytes. In this work, we have determined the percentage of HCV-infected hepatocytes, the histological activity index, and the viremia levels in chronically HCV-infected patients with different grades of liver injury to investigate any possible correlation between them. For that purpose, liver biopsies from 27 patients with HCV chronic hepatitis were analyzed by in situ hybridization. This technique revealed that the percentage of infected hepatocytes ranged from 0.04% to 83.6%. Regarding the viremia levels, HCV RNA concentration ranged from 1.8 x 10(3) to 1.4 x 10(6) genome copies/ml. A significant correlation (r = 0.54; P = 0.003) between the percentage of infected hepatocytes and the viremia levels was found. In contrast, no correlation was observed between the percentage of HCV-infected hepatocytes or the viremia levels and the histological activity index. In conclusion, we have shown that the HCV viremia reflects the extent of the infection in the liver and that the liver injury in chronic HCV infection is not directly related to either the number of infected hepatocytes or the serum HCV RNA concentration.