Effect of anti-viral therapy for occult hepatitis C virus infection

Authors


Dr V. Carreño, C/Guzmán el Bueno, 72, 28015 Madrid, Spain. E-mail: fehvhpa@fehv.org

Summary

Background  Occult hepatitis C virus infection is defined by the presence of hepatitis C virus-RNA in liver but with undetectable anti-hepatitis C virus and serum viral RNA.

Aim  To study the response to anti-viral therapy in occult hepatitis C virus infection to assess the pathogenic effect of occult hepatitis C virus.

Methods  Ten patients with occult hepatitis C virus infection were treated with pegylated-interferon plus ribavirin for 24 weeks and were followed-up 24 weeks after therapy. All patients had abnormal alanine aminotransferase, hepatitis C virus-RNA positive in peripheral blood mononuclear cells and liver necroinflammation.

Results  At the end of treatment and follow-up, the percentage of patients with normal alanine aminotransferase was 80% (95% CI: 48–96%) and 60% (95% CI: 31–84%) respectively, and hepatitis C virus-RNA in peripheral blood mononuclear cells was negative in 80% (95% CI: 48–96%) and 70% (95% CI: 40–90%) cases. At the end of follow-up sustained response was observed in 30% (95% CI: 11–61%) of cases. Five patients underwent a second liver biopsy. In all cases, liver hepatitis C virus-RNA persisted, although hepatitis C virus-RNA load was significantly lower (3.2 × 104 ± 5.1 × 104 copies/μg RNA) than in the basal biopsy (2.4 × 105 ± 3.8 × 105 copies/μg RNA); (P = 0.043). Necroinflammation and fibrosis decreased in three cases.

Conclusion  The biochemical, virological and histological response to therapy achieved in patients with occult hepatitis C virus infection demonstrates the pathologic effects of occult hepatitis C virus.

Introduction

Hepatitis C virus (HCV) is the major cause of chronic hepatitis and hepatic fibrosis that progresses to cirrhosis in 20% of the cases and around 5% will develop hepatocellular carcinoma.1–7.

Recently a new form of HCV infection called occult HCV infection has been described.8 Occult HCV infection is defined by the presence of HCV-RNA in liver cells but with undetectable anti-HCV and serum viral RNA.8 Patients with occult HCV infection have abnormal liver function tests and 35% of them have histological damage, including liver cirrhosis.8 All these facts suggest that occult HCV could have a pathogenic role and may cause liver damage.

Most patients with occult HCV infection have a milder histological lesion than patients with chronic hepatitis C.9 Thus it is not clear whether the cause of the liver damage in these patients is due to the presence of occult HCV. One way to demonstrate the relationship between occult HCV infection and liver disease activity could be to study the biochemical, virological and histological response to anti-viral therapy. If changes in biochemical, virological and histological rates are observed during anti-viral therapy this will strongly support the pathogenic role of occult HCV infection.

In addition, the presence of the genomic and antigenomic HCV-RNA strands has been detected in peripheral blood mononuclear cells (PBMC) of patients with occult HCV infection,9, 10 indicating that HCV is replicating in these cells.

Currently, anti-viral therapy for chronic hepatitis C is successful in approximately 50% of infected patients, resulting in clearance of HCV-RNA from serum, normalization of liver function tests and improvement of liver histology.11, 12 This standard treatment is the combination of pegylated-interferon (1.5 μg/kg subcutaneously once weekly) and ribavirin (800–1200 mg/p.o./day) for 6–12 months.

The aim of this work was to study the response to anti-viral treatment in patients with occult HCV infection in order to assess the possible pathogenic effect of occult HCV.

Materials and methods

Patients

The inclusion criteria in the present work were: patients with occult HCV infection (HCV-RNA in liver by reverse transcriptase-polymerase chain reaction (RT-PCR) and by in situ hybridization in the absence of anti-HCV and serum HCV-RNA), with abnormal alanine aminotransferase (ALT) values for at least 12 months, HCV-RNA positive in PBMC and the presence of necroinflammatory activity in liver biopsy performed within 1 year before the study entry. None of the patients could have received anti-viral therapy before the inclusion in the study.

Among our 57 patients with occult HCV infection described in a previous report,8 20 fulfilled the above mentioned inclusion criteria and were required to receive interferon-based anti-viral therapy. Other causes of liver diseases were excluded in all patients based on analytical, clinical and epidemiological data: infection by hepatitis B virus (hepatitis B surface antigen and serum HBV-DNA negative), HCV (anti-HCV and serum HCV-RNA negative), autoimmunity, metabolic and genetic disorders, alcohol intake, drug toxicity, steatosis, steatohepatitis, etc., and all cases were negative for anti-HIV antibodies. Ten of these 20 patients gave their written informed consent to be included in the protocol. The study was done in accordance with the Declaration of Helsinki.

The patients received standard doses of pegylated-interferon (Peg-Intron; Schering-Plough, Kenilworth, NJ, USA: 1.5 μg/kg/week) plus ribavirin (Rebetol; Schering-Plough: 800–1200 mg/day, depending on the body weight) for 24 weeks and were followed-up monthly during therapy and every 3 months during the post-treatment follow-up (24 weeks). In the study protocol, a second liver biopsy taken at the end of follow-up was planned, and this proposal was accepted by five patients.

Primary efficacy of the treatment was defined as ALT normalization at the end of treatment (biochemical response). The second efficacy endpoint was defined as undetectable HCV-RNA in PBMC (virological response) at the end of treatment. A patient who achieved biochemical and virological response at the end of treatment was considered as a complete responder. Sustained biochemical, virological or complete responders were defined as patients who achieved a biochemical, virological or complete response at the end of treatment and continued to be so at the end of follow-up.

Methods

Before inclusion in the study, serum and PBMC samples were obtained from all the patients to test once again the anti-HCV and HCV-RNA status. In addition, anti-HCV and HCV-RNA were also checked in serum and PBMC at the third month of treatment, at the end of therapy and at the end of the follow-up period. Once PBMC samples were obtained, they were stored in RNALater (Ambion Inc., Austin, TX, USA) at −20 °C until use.

Serological markers

Anti-HCV was determined by a commercial enzyme immunoassay test (INNOTEST HCV Ab IV, Innogenetics, Ghent Belgium).

HCV-RNA detection in serum and PBMC

Total RNA was isolated from serum and PBMC samples as described8 and HCV-RNA was tested by RT-PCR, using primers from the 5′ untranslated region of the HCV genome.8

HCV-RNA detection in liver biopsies

Five patients accepted to undergo a second liver biopsy after the end of the follow-up period (24 weeks after treatment). Liver biopsies were cut into two portions: one portion for histological diagnosis and the other one was embedded (no later than 30 s) in RNALater (Ambion Inc.) and stored at −20 °C until use. Basal and post-treatment liver biopsies were examined by a single central pathologist in a blinded fashion according to the Sheuer score.13

Total RNA was isolated from liver samples using the SV Total RNA Isolation kit (Promega Co., Madison, WI, USA). After precipitation, the pellet was dissolved in diethyl-pyrocarbonate-treated water and total RNA concentration was determined by spectrophotometry. In order to compare intrahepatic HCV-RNA load before and after treatment in these five patients, HCV-RNA quantification was simultaneously performed in a stored aliquot at −80 °C of total RNA isolated from the basal or post-treatment liver biopsies. Quantification of the 5′ non-coding region of HCV-RNA was done by real-time RT-PCR, using the thermostable enzyme Tth (Applied Biosystems, Foster City, CA, USA) for the synthesis of cDNA at high temperature. Real-time PCR was run in a Light Cycler (Roche Molecular Biochemicals, Indianapolis, IN, USA) with 2 μL of cDNA in a final volume of 20 μL, using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals). Standard curve constructed with synthetic HCV-RNA was performed for the quantification of the HCV-RNA. The results were expressed as copies per microgram of total RNA.

To assure the specificity of the results HCV-RNA detection was performed in a blinded fashion in different days by two different operators. In addition, PBMC from 10 healthy anti-HCV negative subjects, total RNA isolated form HepG2 cells and blanks were used as negative controls. Finally, all procedures were performed following the Kwok and Higuchi recommendations.14

In situ hybridization

Paraffin-embedded liver sections (4 μm) were pretreated for in situ hybridization as described.15 HCV-RNA positive strand was detected with a complementary RNA probe obtained by in vitro transcription of the pC5′NCR plasmid, which contains the complete 5′NC region of the HCV genome. Detection of the negative HCV-RNA strand was done with a complementary RNA probe spanning 390 nucleotides of the HCV core coding region, obtained by in vitro transcription of the pCcore plasmid. Hybridization conditions for the in situ detection and specificity controls were as described.15 The percentage of infected cells was determined by visual inspection counting at least 2000 cells from each liver section.

Statistical analysis

The statistical analysis was performed with the SPSS 9.0 for Windows (SPSS, Inc., Chicago, IL, USA). Categorical variables were expressed as the proportion and the 95% confidence interval (CI) proportion. 95% CI of proportions were estimated by the modified Wald method.16 Continuous variables were expressed as the mean and the 95% CI of the mean, except when indicated. ALT levels measured at baseline, at the third month, at the end of therapy and at the end of follow-up were compared with the Friedman test for related samples. Two-by-two comparisons were performed with the non-parametric Wilcoxon Signed Ranks test. All comparisons were performed two-sided and a P-value <0.05 was considered statistically significant.

Results

Basal characteristics of the 10 patients included in the study are shown in Table 1. Anti-HCV and serum HCV-RNA always remained negative in all patients during the whole treatment and follow-up period.

Table 1.  Basal characteristics of the patients included in the study (n = 10)
  1. Continuous variables are expressed as the mean (95% CI of the mean).

  2. BMI, body mass index; ALT, alanine aminotransferase; HCV, hepatitis C virus.

  3. * Expressed as the mean ± s.d.

  4. † According to Scheuer et al.13 P, portal inflammation; L, lobulillar inflammation; F, fibrosis.

Gender (male/female)7/3
Age (years)40.7 (34.1–47.3)
BMI25 (23–27)
Estimated duration of abnormal liver function tests (months)61.7 (39.0–84.4)
ALT (IU/L)74.4 (58.5–90.3)
HCV genotype (1b/other)10/0
Mean % of HCV infected hepatocytes*3.3 ± 2.7
Triglycerides (mg/dL)94.9 (77.8–112.0)
Cholesterol (mg/dL)208.8 (187.0–230.6)
Glucose (mg/dL)93.7 (88.9–98.5)
Iron (μg/dL)94.8 (80.5–109.1)
Histological damage†
 P1 L1 F14
 P1 L1 F03
 P1 L0 F01
 P0 L1 F01
 P2 L2 F21

All patients completed the treatment and the anti-viral therapy was well tolerated and no dose modification was needed. At the third month of treatment, four of 10 patients [40% (95% CI: 12–74%)] normalized ALT levels (ALT ≤43 IU/L). At the end of treatment and at the end of the follow-up period, the percentage of patients with normal ALT values was 80% [8/10 (95% CI: 48–96%)] and 60% [6/10 (95% CI: 31–84%)], respectively (Table 2). Figure 1 shows the evolution of the mean ALT levels from baseline to the end of the follow-up period. In this sense, at the third month of treatment a slight increase was observed in the mean ALT level, although no statistical differences were found when compared with basal value [third month: 79.5 IU/L (95% CI: 8.8–150.2 IU/L) vs. basal: 74.4 IU/L (95% CI: 58.5–90.3 IU/L); P = 0.169]. This slight increase was due to two patients who presented an increase of ALT levels when compared with basal sample (from 77 to 107 IU/L and from 46 to 350 IU/L, respectively). At the sixth month (end of treatment) the mean ALT level was lower in comparison with the basal one but without statistical differences [sixth month: 52.5 IU/L (95% CI: 11.7–93.3 IU/L) vs. basal; P = 0.074]. However, at the end of the follow-up period ALT levels were significantly lower than basal values [end of follow-up: 36.9 IU/L (95% CI: 26.8–47.0 IU/L) vs. basal; P = 0.007].

Table 2.  Alanine aminotransferase (ALT) levels and hepatitis C virus (HCV)-RNA detection in peripheral blood mononuclear cells (PBMC) at baseline, at the end of treatment and at the end of follow-up of all patients included in the study
Patient no.BasalFinal treatmentFinal follow-up
ALT (IU/L)HCV-RNA in PBMCALT (IU/L)HCV-RNA in PBMCALT (IU/L)HCV-RNA in PBMC
  1. Normal ALT ≤ 43 IU/L.

175Pos24Neg30Pos
277Pos43Pos34Neg
364Pos27Neg21Neg
470Pos39Neg20Neg
555Pos25Neg46Pos
697Pos39Neg29Neg
797Pos68Neg55Neg
8113Pos31Neg58Neg
950Pos19Pos26Pos
1046Pos210Neg50Neg
Figure 1.

Box-plot representations of the evolution of the alanine aminotransferase (ALT) levels from the baseline to the end of follow-up. Each box is drawn from the lower quartile (Q1) to the upper quartile (Q3) and the horizontal lines across the boxes indicate the median. The whiskers are drawn from the Q3 to the maximum and from the Q1 to the minimum. The P-value showed in the figure was obtained using the Friedman test for the comparison of four related samples. Outliers at 3 months of therapy (350 IU/L) and at the end of therapy (210 IU/L), corresponding to the ALT values of the same patient, are not represented in the figure.

Regarding HCV-RNA status in PBMC, six of 10 patients [60% (95% CI: 31–84%)] were negative at the third month of treatment. At the end of treatment, eight of 10 [80% (95% CI: 48–96%)] patients were HCV-RNA negative in PBMC. Finally, the percentage of patients with undetectable HCV-RNA in PBMC at the end of follow-up was 70% [7/10 (95% CI: 40–90%)] (Table 2).

Overall efficacy results are shown in Table 3.

Table 3.  Overall results on the efficacy of the treatment
 End of treatmentEnd of follow-up (sustained response)
  1. Data are expressed as the percentage (95% CI of the percentage).

Biochemical response8/10 [80% (48–96%)]6/10 [60% (31–84%)]
Virological response8/10 [80% (48–96%)]6/10 [60% (31–84%)]
Complete response6/10 [60% (31–84%)]3/10 [30% (11–61%)]

In the second liver biopsy performed in five patients (two complete responders, one virological responder, one biochemical responder and one non-responder) it was found that HCV-RNA persisted in liver cells in all cases, detected by real time RT-PCR (Table 4). However, mean HCV-RNA load in liver was significantly lower in the post-treatment biopsy than in the basal one (mean ± s.d.: 3.2 × 104 ± 5.1 × 104 copies/μg total RNA vs. 2.4 × 105 ± 3.8 × 105 copies/μg total RNA, P = 0.043) (Table 4). Percentage of infected hepatocytes was lower in the final biopsy (mean ± s.d.: 2.2 ± 1.9%) compared with the basal biopsy (mean ± s.d.: 3.5 ± 1.6%), although the differences did not reach statistical significance (P = 0.225). Regarding liver histology, two patients (one complete responder and one biochemical responder) remained unchanged while in the other three cases (one biochemical responder, one complete responder and one non-responder) necroinflammatory activity and fibrosis score decreased (Table 4).

Table 4.  Biochemical, virological and histological data from the five patients who underwent a second liver biopsy
Patient no.Pre-treatment: baselinePost-treatment: end of follow-up
ALT (IU/L)HCV-RNA in PBMCHistological damageIntrahepatic HCV-RNA (copies/μg RNA)ALT (IU/L)HCV-RNA in PBMCHistological damageIntrahepatic HCV-RNA (copies/μg RNA)
  1. Normal ALT ≤43 IU/L.

  2. Histological damage: portal inflammation (P); lobulillar inflammation (L); fibrosis (F).

  3. ALT, alanine aminotransferase; HCV, hepatitis C virus; PBMC, peripheral blood mononuclear cells.

175PosP1 L1 F11.5 × 10430PosP0 L0 F01.2 × 103
277PosP1 L1 F01.2 × 10434NegP1 L1 F02.4 × 103
470PosP2 L2 F29.0 × 10520NegP0 L1 F01.2 × 105
555PosP1 L1 F11.2 × 10446PosP0 L0 F02.2 × 103
797PosP1 L1 F12.6 × 10555NegP1 L1 F13.2 × 104

Discussion

Occult HCV infection is a new form of HCV infection characterized by the presence of HCV-RNA in liver and PBMC in the absence of anti-HCV and HCV-RNA in serum.8, 10 Although patients with occult HCV have liver damage, the relationship between the presence of HCV and the liver damage needs to be determined. One way to demonstrate this is to assess the response to anti-viral therapy in patients with occult HCV infection. Thus, in this work we have treated 10 patients with occult HCV infection with standard doses of pegylated-interferon and ribavirin for 6 months to further confirm this issue.

At the end of treatment, 80% of patients had normal ALT levels and 80% were HCV-RNA negative in PBMC. At the end of follow-up, 30% patients were sustained complete responders, thus, they had normal ALT levels and were HCV-RNA negative in PBMC. In addition, in the five patients with a second liver biopsy, the concentration of HCV-RNA in liver was significantly lower in the post-treatment sample than in the basal biopsy, and the percentage of infected hepatocytes tended to be lower in the post-treatment liver sample than in the basal one. All these data confirm the pathogenic role of occult HCV. These results are similar to those observed in chronic hepatitis C as anti-viral treatment is able to improve the activity of the disease.4, 17, 18

All the patients included in our study were infected by HCV genotype 1b and the currently accepted duration of pegylated-interferon plus ribavirin therapy for patients with genotype 1b is 12 months.19 However, as our patients were HCV-RNA negative in serum and the percentage of infected hepatocytes is lower in patients with occult HCV infection than in chronically infected patients,9 we decided to treat them for 6 months. In support of this schedule, some authors recommend that patients infected with HCV genotype 1b but with serum viral load below 800 000 IU/mL should be treated for 6 months instead of 12 months.20

Hepatitis C virus-RNA remained positive in PBMC from 20% of patients with normal ALT levels after the follow-up. Similar results have been found by other authors, who have reported that HCV-RNA in PBMC can be detected for a mean period of 64 months in chronic hepatitis C patients who responded to interferon therapy.21 Also, Pham et al.22 reported that HCV-RNA can persist in PBMC up to 5 years after spontaneous or treatment-induced recovery of HCV infection. Regarding HCV-RNA in liver, we have found that HCV-RNA remained positive in the five patients (two complete responders, one virological responder, one biochemical responder and one non-responder) with available post-treatment liver biopsies. This finding is in agreement with a previous work,21 in which it has been reported that HCV-RNA persists in the liver of 27% of chronic hepatitis C patients with sustained response for years after the end of the therapy. Thus, HCV-RNA persistence in liver and/or PBMC may be relevant because reactivation of the disease may take place. In this sense, reactivation of the disease after immunosuppression in a patient with chronic hepatitis C who was complete responder, with serum HCV-RNA negative and normal ALT has been described.23 In our study, histological improvement was found in three of the five second liver biopsies. Although the number of paired liver biopsies analysed in this study is small, all these results suggest that interferon-based anti-viral therapy may have potential benefits in the treatment of patients with occult HCV infection. However, these results should be confirmed in future studies with a larger number of patients.

In summary, biochemical, virological and histological response to the anti-viral therapy obtained in patients with occult HCV infection demonstrates the pathogenic effect of this novel form of HCV infection.

Acknowledgements

This work has been supported by the Fundación Investigaciones Biomédicas.

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