Juan Manuel López-Alcorocho

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.

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.

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.

Detection of TT Virus DNA in Liver Biopsies by in Situ Hybridization

A novel hepatitis-associated virus named TT virus (TTV) has been isolated. However, its hepatotropism has not been proven. We have retrospectively analyzed the presence of TTV-DNA by polymerase chain reaction (PCR) and in situ hybridization in liver biopsies from 30 patients with liver disease (15 TTV-DNA-positive and 15 TTV-DNA-negative in serum), and prospectively in serum and liver from eight patients with normal liver histology. TTV-DNA was detected by PCR in the liver from the 15 patients with serum TTV-DNA and in serum and liver of two of the eight patients without liver disease. TTV-DNA titers in liver were 10 times higher than in serum, although no correlation between TTV-DNA titers in serum and liver were observed. In situ hybridization shows positive signals in the hepatocytes of the 17 patients infected by TTV but in none of the TTV-DNA-negative patients by PCR. No morphological changes were observed in the hepatocytes showing hybridization signals. The percentage of positive hepatocytes ranged from 2.1% to 30% and correlated with the TTV-DNA titers in liver (r = 0.54; P = 0.037). In conclusion, our results show that TTV is able to infect liver cells although they do not support a role for TTV in causing liver disease.

Comparative study between occult hepatitis C virus infection and chronic hepatitis C.

Summary.  We have recently described the presence of occult hepatitis C virus (HCV) infection (HCV‐RNA in liver in the absence of anti‐HCV and serum HCV‐RNA) in patients with persistently abnormal liver function tests of unknown aetiology. The aim of this study was to compare the characteristics of patients with occult HCV infection vs those of patients with chronic hepatitis C. We compared clinical features of 68 patients with occult HCV infection and 69 untreated chronic HCV patients (anti‐HCV and serum HCV‐RNA positive), matched for age, gender, duration of abnormal liver function tests and body mass index. Aspartate aminotransferase and alanine aminotransferase were higher (P < 0.001) in chronic HCV, but cholesterol and triglycerides were significantly higher in patients with occult HCV infection (P < 0.001 and P = 0.002). Chronic HCV patients had higher gamma‐globulin (P = 0.005), alpha‐foetoprotein (P < 0.001) and iron (P < 0.001) levels. Percentage of patients with necroinflammatory activity and fibrosis was higher (P < 0.001) in chronic HCV than in occult HCV infection. Mean percentage of infected hepatocytes was higher (P = 0.001) in chronic HCV (10.1%) than in occult HCV infection (5.3%). This occult HCV infection is a milder disease than chronic HCV, and this could be related to the significantly lower number of infected hepatocytes observed in occult HCV.

Hepatitis C Virus (HCV) and Hepatitis B Virus (HBV) Can Coinfect the Same Hepatocyte in the Liver of Patients with Chronic HCV and Occult HBV Infection

ABSTRACT In this work, we have shown that hepatitis C virus (HCV) and hepatitis B virus (HBV) can coexist in the same hepatocyte using double fluorescent in situ hybridization in liver biopsy samples from patients with chronic HCV infection with occult HBV infection. Digital image analysis of hybridization signals showed that the HBV DNA levels in coinfected hepatocytes were lower than those in cells infected only with HBV. This finding supports the hypothesis of inhibition of HBV replication by HCV. Furthermore, HCV RNA levels were lower in coinfected cells than in cells infected only with HCV, suggesting that HBV may also inhibit HCV replication.

Prevalence of hepatitis B, hepatitis C, GB virus C/hepatitis G and TT viruses in predialysis and hemodialysis patients.

Patients with chronic renal failure on hemodialysis have a high risk of infections with viruses such as hepatitis B (HBV), hepatitis C (HCV), GB virus C/hepatitis G (GBV‐C/HGV) and TT (TTV) viruses. The prevalence of HBV, HCV, GBV‐C/HGV and TTV in patients with chronic renal failure who are on conservative management before entering into a hemodialysis program (predialysis) in comparison with hemodialyzed patients was studied to elucidate whether the high prevalence of these viruses is influenced by that observed in the predialysis stage. The presence of hepatitis B virus surface antigen (HBsAg), HCV RNA, GBV‐C/HGV RNA and TTV DNA was analyzed in sera from 80 patients with chronic renal failure (35 on predialysis and 45 on hemodialysis). HBsAg, HCV RNA, GBV‐C/HGV RNA and TTV DNA were detected in one (2.8%), six (17.1%), eight (22.5%) and 16 (45.7%) of the 35 patients on predialysis. Two (5.7%) of these patients were coinfected with HCV and GBV‐C/HGV, whereas six (17.1%) had GBV‐C/HGV and TTV coinfection. In the 45 hemodialyzed patients, HBsAg, HCV RNA, GBV‐C/HGV RNA and TTV DNA were detected in one (2.2%), two (4.4%), seven (15.5%) and 26 (57.7%). One (2.2%) patient had HBV and TTV coinfection, two (4.4%) HCV and TTV coinfection whereas four (8.8%) were coinfected with GBV‐C/HGV and TTV. No differences regarding age, gender, previous surgery and number of transfusions were found between infected and uninfected patients within and between both groups. In conclusion, the prevalence of the viruses studied in predialysis may influence their prevalence in dialysis units. J. Med. Virol. 63:103–107, 2001.

Existence of Distinct GB Virus C/Hepatitis G Virus Variants with Different Tropism

ABSTRACT To study the existence of GB virus C/hepatitis G virus (GBV-C/HGV) variants with different tropism, we have analyzed the heterogeneity and quasispecies composition of GBV-C/HGV isolated from in vitro-infected peripheral blood mononuclear cells (PBMC) and from sera, livers, and PBMC from two chronically infected patients. For this purpose, the GBV-C/HGV 5′ noncoding region (5′NCR) was amplified by reverse transcription-PCR and the amplified products were cloned and sequenced. These analyses showed that the master 5′NCR sequences isolated from the in vitro-infected PBMC and from the PBMC isolated from the patient whose serum was used as the inoculum were identical but different from that of the inoculum. Furthermore, phylogenetic analysis revealed that all PBMC sequences grouped together into a branch which was separate from those of the inoculum. For one of the two chronically infected patients, all the sequences from the PBMC and one from the liver clustered into a single branch while the sequences from the serum and all the other liver sequences grouped together in the other branch. For the other patient, the sequences from the serum and PBMC and three sequences from the liver grouped together into one branch, while the remaining five sequences from the liver were separated in a different cluster. In conclusion, our results support the existence of different GBV-C/HGV variants with different tissue tropism.

Percentage of Hepatitis C Virus-Infected Hepatocytes Is a Better Predictor of Response Than Serum Viremia Levels

Pegylated alpha-interferon plus ribavirin is the current therapy for chronic hepatitis C virus (HCV) infection. Serum HCV-RNA concentration before treatment has been identified as an independent predictive factor of response. We have compared the percentage of HCV-infected hepatocytes with the concentration of serum HCV-RNA in baseline samples as predictors of response. We included 97 patients with chronic HCV infection (genotype 1), treated with pegylated-interferon-alpha2b plus ribavirin. Of these 97, 38 (39%) were sustained responders and 59 (61%) were not. Statistical differences between responders and nonresponders were found regarding the percentage of infected hepatocytes (6.83+/-4.50% versus 13.44+/-10.05%; P=0.00003) but not in serum HCV-RNA concentration [1.71+/-2.70 (x10(6) IU/L) versus 1.32+/-1.86 (x10(6) IU/L); P=0.40694]. Other factors associated with response were age, gamma-glutamyl transpeptidase level, and absence of previous therapy. Logistic regression demonstrated that percentage of infected hepatocytes (odds ratio, 1.160; 95% confidence interval, 1.065-1.264) and previous therapy (odds ratio, 0.294; 95% confidence interval, 0.109-0.795) were significant predictive factors for response. Therefore, the percentage of infected hepatocytes in liver biopsy before treatment is a better predictive factor of sustained response to 48 weeks of therapy with pegylated alpha-interferon plus ribavirin than serum HCV-RNA concentration in baseline serum sample.