Low virological response to interferon in children with chronic hepatitis C

Journal offiepafolog)~ 1999;31: 60461

I

Printed in Denrnurk All rights reserved

Munksguurd Copenhu~rn

Journal of Hepatology ISSN 0168-8278

Low virological response to intederon in children with chronic hepatitis C Pasqualina

Pensati, Raffaele

Department of Pediatriu,

Iorio, Salvatore Botta, Concetta Tuccillo’, Sara Donetto, Giuseppina Ciarlo and Angela Vegnente

University of Naples “Federico II”,

Pietro Vajro,

‘Departmentof Internal Medicine “F: Mograssi”, Second University (?f Nuples, Italy

Background/Aims: Children with chronic hepatitis C were recently found to have higher rates of sustained response to interferon compared to adults. The aim of this study was to verify the response to interferon using frequent viremia measurements. Methods: Sera from 25 children (13 males; mean age 7.9 years) with chronic hepatitis C, treated with recombinant alpha-2b interferon for 12 months, were tested for liver function tests and viremia levels for a median of 27.5 months. Autoantibodies were evaluated during and after interferon. Results: Fifteen patients completed 12 months of interferon; treatment was stopped in 10 other patients. In 11 (44%) patients viremia was undetectable already at the second administration of interferon; one of them remained viremia-free up to the end of follow-up and had persistently normal alanine-aminotransferase levels (complete sustained responder). A

complete sustained response was observed only in one other patient, who normalized alanine aminotransferase and cleared viremia from the 3rd month of therapy. Three patients with persistent viremia normalized alanine-aminotransferase from the 3rd week of therapy up to the end of follow-up (biochemical sustained responders). Viremia was undetectable during treatment in four patients, who stopped interferon because of worsening in hypertransaminasemia. Three of these four patients were anti-liver-kidney microsomal type l-positive. Conclusions: In this study the response rate to interferon was very low and viremia and transaminase findings were often discordant.

C

of adult patients with normal ALT serum levels after treatment with IFN (11,12). In recent studies, IFN was more effective in children than in adults in terms of ALT normalization and HCV RNA clearance (I 3-l 8). In most of these studies the virological response to IFN was evaluated on the basis of sporadic determinations of serum HCV RNA (13,15,16,18). However, a body of evidence suggests that HCV RNA level is not a constant, but varies spontaneously or as a consequence of endogenous factors such as the magnitude of the immune response, medications such as corticosteroids and differences in serum sample preparation for HCV RNA determination (18-23). Therefore, frequent quantitations of viremia during IFN therapy could provide a better measure of the response. Some pre-treatment characteristics in adults have been associated with a greater likelihood of response to a-INF; the most important are low basal levels of

the only approved therapy for chronic hepatitis C is alpha-interferon ((x-INF) (l--3). Controlled studies in adults with chronic hepatitis C (CHC) have shown that a standard regimen of or-INF therapy induced normalization of alanine-aminotransferase (ALT) serum levels during treatment in 35.-50% of the patients. Unfortunately, 50% to 80% of biochemical responders relapsed within 6 months of discontinuation of therapy (4-9). With the advent of HCV genome tests, it became apparent that detectable HCV RNA in serum is a better indicator of response (10). However, HCV RNA has been found in 10-40%1 URRJZNTLY,

Received 9 Fehruqy; revised I9 April; accepted 26 April I999

Correspondence: Pasqualina Pensati, Dipartimento di Pediatria, UniversitB di Napoli “Federico II” Via Sergio Pansini n. 5 80131 Napoli, Italy. Tel: 39 81 746 3500. Fax: 39 81 545 1278. e-mail: [email protected]

604

Key WV& Children; Chronic hepatitis; HCV viral load; Hepatitis C virus; Interferon alfa; Viral clearance.

IFN treatment in children with chronic hepatitis C

serum HCV RNA, the absence of cirrhosis on the pretreatment liver biopsy specimen, and the presence of a genotype other than lb (24-26). To our knowledge, there is a lack of exhaustive data about basal viral load and its fluctuations during and after IFN treatment in children with CHC. We measured HCV RNA levels, using frequent quantitations of viremia, in 25 children with CHC, treated with ol-IFN for 12 months. The aim of our study was to obtain a more accurate definition of end-of-treatment response and of sustained response (after 18 months of posttherapy follow-up). In addition, because IFN therapy is expensive, long-lasting and associated with several, and sometimes severe, side effects (27,28), we also attempted to identify parameters, particularly virologic parameters such as HCV RNA levels and genotype, that could predict the response to IFN, in order to better select patients who could benefit from the treatment.

Materials and Methods Twenty-five consecutive patients (13 males; mean age 7.7 years; range 2.8-14.5 years) with CHC, attending the Liver Unit of the Department of Pediatrics of the University of Naples “Federico II”, were included in a prospective study lasting from November 1995 to June 1998. The patients’ basal characteristics are listed in Table 1.

TABLE

1

Basal characteristics Male/Female/Median

of 25 patients age

Source of infection: Blood transfusion Vertical transmission Minor surgery Minor parenteral exposure Unknown Median duration of HCV infection Median ALT serum levels Median HCV RNA serum levels

with CHC

Diagnosis Chronic hepatitis C was diagnosed on the basis of elevated serum ALT levels (1.5 times above the normal range on at least two occasions) over 6 months, anti-HCV antibodies revealed by a third-generation recombinant immunoblot assay (Ortho Diagnostic System, Raritan, NY, USA), serum HCV RNA and histological evidence of chronic hepatitis. Liver biopsy was performed in all patients within the 6 months before enrollment in the study, and liver histology was evaluated according to the criteria of Scheuer & Desmet et al. (29,30). Causes of chronic liver disease other than HCV were excluded in all patients. None of the patients showed serological evidence of hepatitis B virus (HBV), cytomegalovirus or human immunodeficiency virus infection. Serum values of alpha-l antitrypsin were within the normal range. Autoimmune hepatitis was ruled out according to the criteria established by the International Autoimmune Hepatitis Group (31). Hemochromatosis was excluded on the basis of normal serum concentrations of iron, transferrin and ferritin. Ceruloplasmin serum concentration and urinary copper excretion were normal, thus ruling out Wilson’s disease. No patient had clinical and/or laboratory evidence of other more common inborn errors of metabolism associated with hypertransaminasemia, such as galactosemia, hereditary fructose intolerance, glycogen storage disease, ornithine carbamyl transferase deficiency, tyrosinemia, mitochondrial cytopathies, Gaucher’s disease, and Niemann-Pick disease. No patient had evidence of drug- or toxin-induced liver injury. Patients with cirrhosis or chronic systemic diseases were also excluded. At the beginning of IFN treatment all the enrolled patients were asymptomatic and none had a history of acute liver injury, malignancy, or prior use of IFN preparations.

IFN therapy Patients received recombinant a-2b IFN at the dosage of 5 million units (MU) per square meter of body surface, intramuscularly, three times a week for 12 months. According to previous studies (l&32,33), treatment was discontinued in case of severe side effects, including increases of ALT values to more than 6 times the upper reference limit, or because ALT levels did not decrease after 6 months of treatment. Interferon discontinuation was not based on viremia because HCV RNA serum levels were determined at the end of the study in all patients.

13/12/7.7 years (range 2.8-14.5) No. pts (%) 10 (40) ll(44) 2 (8) 1 (4) 1 (4) 3.4 years (range 0.5-l 1 .l) 89 U/l (range 62-200) 3.587X 10^6 Eq/ml (range 0.202-21.395)

Genotype: la lb 2a 3a 2a+lb 2a+2c

no. pts (%)

Histological features: Minimal chronic hepatitis Mild chronic hepatitis Moderate chronic hepatitis Severe chronic hepatitis HCV positivity in parents: Mother Mother and father None Unknown

no. pts (%)

6 (24) 13 (52) 3 1 1 1

(12) (4) (4) (4)

4 (16) 15 (60) 6 (24) None (0) no. pts (%) 7 (28) 4 (16) 13 (52) 1 (4)

Follow-up schedule Blood samples for serum HCV RNA and ALT determinations were collected at the following times: 2 weeks and 1 week before IFN therapy, weekly for the first 3 weeks of treatment, monthly until the 6th month of therapy, at the 9th and 12th month of therapy, and, thereafter, at the lst, 3rd, 6th, 12th and 18th month of post-therapy followup. On the same occasions, clinical evaluation, hematological tests and the usual liver function tests were also performed. In all patients, serum HCV RNA determinations were performed at the end of follow-up. The baseline serum HCV RNA concentration for each patient was calculated as the mean of the HCV RNA concentration determined at weeks -2 and - 1. Because both CHC and IFN treatment may be associated with autoimmunity, cryoglobulinemia and thyroid diseases (3438), all patients were tested, before enrollment, every 3 months during therapy and every 6 months after therapy, for antinuclear (ANA), anti-smooth muscle (SMA), anti-liver-kidney microsomal type 1 (LKM-1) and cryoglobulins, total anti-gastric parietal cell (GPC) autoantibodies, thyroxine (T4), free thyroxine (fT4), total tri-iodothyronine (T3), free tri-iodothyronine (ff3), and thyroid stimulating hormone (TSH).

Approval Each patient’s parent gave written informed consent to the study. The study was approved by our Hospital Ethics Committee. The Declaration of Helsinki on experimentation in humans was observed in all aspects of this study.

Virological studies Blood samples for serum HCV RNA determination were centrifuged within 2 h of collection and immediately separated. Serum samples

605

P. Pensati TABLE

et al.

2

Type of response

to IFN therapy

at the end of treatment

and during

End of IFN therapy 1st month post-therapy follow-up 6th month post-therapy follow-up 12th month post-therapy follow-up 18th month post-therapy follow-up Values are given as number of patients with different * 2 of these patients became nonresponders. # 1 of these patients became a biochemical responder.

post-therapy

follow-up

in 25 children

with CHC

Complete response

Virological response

Biochemical response

No response

3 (12%) 3# (12%) 2 (8%) 2 (8%) 2 (8%)

0 0 0 0 0

4* (16%) 2 (8%) 3 ( 12%) 3 ( 12%) 3 (12%)

18 20 20 20 20

(56%) (80%) (80%) (80%) (80%)

type of response

end of treatment. Complete sustained response was defined as the persistence of this response until the end of the post-therapy followup. The occurrence, during treatment, of undetectable HCV RNA by RT-PCR or serum ALT normalization, persisting until the end of therapy, was considered to be a virologicul and a biochemical end-of’ treatment response, respectively. The same conditions persisting until the end of post-therapy follow-up were considered to be a virological and a biochemical sustnined response, respectively. The term relupsc was used to describe an increase of ALT serum levels or the appearance of HCV RNA after a biochemical or virological end-of-treatment response.

were stored in aliquots at -80” C until the analysis was performed. The HCV RNA titer was measured with the most recent version of the branched-chain DNA (bDNA) assay (Quantiplex HCV-RNA 2.0 Assay, Chiron Corporation Emeryville, CA, USA). This version is more sensitive than version 1.0, having a sensitivity limit of approximately 200 000 equivalent genomes/ml. In addition, it is less dependent on HCV genetic variability and provides a more accurate quantitation of genotypes 2 and 3 (39). In patients with serum HCV RNA levels below the bDNA 2.0 assay detection threshold, a commercially available reverse trancriptase polymerase chain reaction (RTPCR) method (Amplicor, Roche Molecular Diagnostic System, Branchburg. NJ, USA) was used to detect serum HCV RNA. This method has a sensitivity limit of about 100 copies/ml. Genotypes were determined by reverse hybridization with a probe hybridization assay (InnoLiPA HCV Innogenetics, Ghent, Belgium) that has been shown to accurately identify HCV genotypes 1 through 6, according to the classification proposed by Simmonds et al. (40).

IFN side ejJect.7 All patients and their parents were invited to record on a diary-card the side effects that occurred during IFN therapy; cards were collected at each consultation. Evaluation and management of IFN-related adverse reactions were as previously reported (28). Statistical analysis Results were expressed as median and range unless indicated otherwise. The Mann-Whitney U and Fisher exact tests were used for statistical analysis. In all cases, a p-value co.05 indicated statistical significance.

Criteriafor response The criteria for response are according to Lindsay (7). Complete endof-treatment response was defined as normalization of serum ALT and undetectable serum HCV RNA by RTPCR during and up to the

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’ Months of post-therapy i

Weeks of IFN therapy

follow-up

No. of patients tested at each control:

606

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25

25

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25

25

23

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20

15

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Fig. 1. Distribution of 25 patients with CHC throughout the observation uccording to HCV RNA und ALT proJiles. q Undetectuble HCV RNA; q ALT normal; n Undetectable HCV RNA and ALT normal. HCV RNA levels were measured by a bDNA assay and RTPCR. The number of’ putients tested decreased between the 4th and 12th month of IFN becuuse IFN wus cc?thdrawn in IO putients.

IFN treatment in children with chronic hepatitis C

Fig. 2. HCV RNA and ALT projiles of 15 patients who completed the course of IFN therapy. l indicates detectable HCV RNA and ALT>40 IUll; W indicates undetectable HCV RNA and ALT>IO IUll; A indicates detectable HCV RNA and ALT
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Results Twenty-five patients were studied for a median of 27.5 months (range 21.5-30.5): 2 weeks before therapy, during IFN therapy and 18 months of post-therapy follow-up. Fifteen (60%) of the 25 patients completed 12 months of treatment, whereas treatment was suspended in ten (40%) patients. Interferon was discontinued at the 6th month in five (20%) patients in whom ALT levels did not decrease after 6 months of therapy, at the 4th month in three (12%) patients who had a gradual increase of serum ALT up to 6 times the normal upper value, and at the third month in two (8%) patients who showed a steep and marked rise of serum

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ALT, with peaks of 7 and 10 times the normal upper value, respectively. The rate of biochemical, virological, and complete response at the end of IFN treatment and during post therapy follow-up is reported in Table 2. During the 1st week of treatment, serum HCV RNA levels decreased in all patients with respect to basal values. Fig. 1 shows the distribution of the 25 patients according to HCV RNA and ALT serum levels throughout the study. The highest number of patients with undetectable HCV RNA occurred at the 3rd week of therapy, and the highest number of patients with normal ALT at the 4th month of therapy. In 11 (44%)

TABLE 3 Genotypes and basal viremia levels in relation to response to IFN At the end of post-therapy follow up

At the end of IFN therapy Complete response

Biochemical response

No response

Complete response

Biochemical response

No response

Genotypes: la lb 2a 3a 2a+lb 2a+2c

1 2 _ _ _

_ 3 1 _ _

5 10 _ 1 1 1

1 _ 1 _ _ _

_ 1 2 _ _

5 12

Mean basal serum HCV RNA (range)

0.253x lo6 Eq/ml* (0.2-0.29)

0.633x lo6 Eq/ml* (0.28-1.57)

5.313X106 Eq/ml* (0.2c21.39)

0.280X lo6 Eqlml (0.24-0.28)

0.700x 106 Eqlml (0.20-1.56)

4.351x106 Eq/ml (0.2c21.39)

Values are given as number of patients for each genotype. * A si@icant difference between basal viremia levels of responders (complete+biochemical) Whitney U).

1 1 1

and nonresponders

was found: ~~0.03 (Mann-

607

P. Pensati et al.

1000

HCV-RNA

xl C% Eq/d

/

L la

lb

2a

3a

2a+zc

Ib+2a

Genotype

Fig. 3. Basal HCV viremia in 25 patients according to HCV genotype and type of response to IFN therupy. A nonresponder. 0 c-SR. 0 h-SR.

patients, serum HCV RNA levels were below the detection limit of the tests at the second administration of IFN (1st week of treatment); one of them remained viremia-free up to the end of follow-up and had persistently normal ALT (complete sustained responder). In the remaining 10 patients the viral load increased from the 3rd to the 6th month of therapy or during the post-therapy follow-up. The profiles of viremia and transaminases in the 15 patients who completed the course of therapy are reported in Fig. 2. One of three complete responders at the end of therapy had a virological relapse at the 6th month of post-therapy follow-up, but maintained normal serum ALT, and was thus classified as a biochemical sustained responder. The remaining two patients maintained a complete response until the end of posttherapy follow-up, and were therefore classified as complete sustained responders. Two of the four biochemical end-of-treatment responders became nonresponders because of a biochemical relapse at the first month of post-therapy follow-up. In four (16%) of the ten patients who stopped IFN, serum HCV RNA was undetectable at the 1st week of treatment and up to treatment withdrawal. One of these four patients stopped IFN at the 6th month because of persistence of hypertransaminasemia. The remaining three patients stopped IFN at the 4th month (one patient) and at the 3rd month (two patients) because of worsening of hypertransaminasemia. All these four patients had a virological relapse during the posttherapy follow-up. A normal ALT level at the 3rd week was a significantly predictive factor of IFN response both at the end of therapy (p=O.O02, Fisher’s exact test) and at the end of follow-up Cp=O.Ol, Fisher’s exact test). In fact, it occurred in six of seven patients who were responders 608

(three complete and four biochemical) at the end of IFN therapy and in four of five patients who were responders at the end of follow-up (two complete and three biochemical). Table 3 shows the correlation between baseline viremia, genotype and response to IFN. None of the responders had basal viremia levels above 1.5X lo6 Eq/ ml. At the end of therapy, a biochemical or complete response was obtained in 86% of patients with basal HCV RNA < 1.0X 10” Eq/ml and in 14% of patients with basal HCV RNA >l.OX lo6 Eq/ml @=0.02, Fisher’s exact test). Fig. 3 shows basal viremia levels according to HCV genotype and response to IFN at the end of follow-up. No significant difference was found between responders and nonresponders for age, sex, route of infection and histological liver damage. Twenty (80%) patients were positive for at least one autoantibody at titers ranging from 1: 10 to 1:80, and the most frequent autoantibody was SMA (Table 3). Interferon treatment was stopped because of transaminase elevation in one SMA-positive, in one ANA-positive and in three of the four LKM-l-positive patients. In these three LKM-l-positive patients the increase in ALT was constantly associated with undetectable serum HCV RNA during the therapy Their LKM-1 titers remained stable at 1:10, 1:40 and 1:80, respectively, during the peak of transaminase and during the post-therapy follow-up. The 4th LKM-l-positive child was one of the two complete sustained responders and she had an LKM-1 titer of 1:lO. Reactivity to cytochrome P4502D6 (CYP2D6) was detected in all four LKM-l-positive cases using an eukaryotically-expressed CYP2D6 in a radioligand immunoassay (41). There were significant differences in the prevalence of ANA positivity before and during treatment (Table 4). However, none of the patients with autoantibody posi-

TABLE

4

Autoantibody IFN therapy

prevalence

in 25 children

with CHC before and during

Autoantibodies

Before IFN therapy

During therapy

ANA SMA LKM GPC Absence of autoantibodies

2 17 4 5 4

7 20 4 4 1

IFN

p-value*

0.027 NS NS NS NS

Values are given as number of patients with autoantibodies. Before IFN therapy, five patients had 2 autoantibodies and one patient 3 autoantibodies. During IFN therapy, eight patients had 2 autoantibodies. * Fisher’s Exact Test.

IFN treatment in children with chronic hepatitis C

tivity fulfilled the score criteria for a diagnosis of “definite” or “probable” autoimmune hepatitis (31). A transitory influenza-like syndrome, characterized by fever, chills, myalgia and arthralgia, occurred in all patients soon after starting treatment. The most frequent side effects were anorexia, abdominal pain, headache, myalgia, vomiting, and irritability All patients experienced at least one adverse reaction to IFN treatment; 80% had more than 5 side effects. In two children, who experienced a severe reaction (febrile seizure and relapsing episodes of epistaxis, respectively), IFN had been stopped at the 4th month of therapy, also because of increases in ALT serum levels up to 6 times the normal upper value. In seven children (28%) the neutrophil count fell below lOOO/mmc and promptly increased when IFN was temporarily discontinued. There was no evidence of cryoglobulinemia and thyroid disorders in any patient.

Discussion This study has brought to light new aspects of the efficacy of IFN therapy in children with CHC. Only 8% of treated children of our study had a complete sustained response at the 18th month of post-therapy follow-up versus 3340% recently reported in other pediatric studies (17,18). This discrepancy is probably attributable to two factors: the virological response in other studies was evaluated on the basis of sporadic determinations of HCV RNA serum levels; and the very strict response criteria used in our study. If we had considered HCV RNA serum levels only at the end of treatment and at the end of post-therapy follow-up, the rate of complete response would have been 20% and 12%, respectively. Our data are consistent with a recent international randomized controlled trial, in which 1071 adults with CHC were treated with different types of IFN (42). In the latter study the rate of sustained response (at the 12th month of post-therapy follow-up) was 6.7% in patients treated with IFN-a-2b. Our data also show that a biochemical response does not correspond to a virological response. In fact, at the end of therapy, only three of seven patients, and at the end of post-therapy follow-up, only two of five patients with persistent ALT normalization had cleared HCV RNA. Consequently, persistent ALT normalization is not invariably associated with a virological response. Moreover, in four patients in whom IFN was withdrawn because of persistence or worsening of hypertransaminasemia, HCV RNA was undetectable throughout the treatment period. In our study, a biochemical response was more frequent than a virological response: 8% of patients had a complete sustained response and 20% had long-term

ALT normalization. A weaker histological activity, a lower fibrosis score and a slower progression rate of fibrosis have been found in HCV-infected adults with normal ALT after IFN therapy than in HCV patients with elevated ALT (43). Thus, although the desirable end-point of IFN treatment is HCV RNA clearance, a long-lasting biochemical response is an alternative, practically feasible goal. Although the main aim of this study was the evaluation of viremia during IFN therapy, the assessment of histological changes could be interesting. On the other hand, since a follow-up biopsy in the present study was scheduled 2 years after the completion of IFN therapy, histological findings are not available. In five children, IFN therapy was associated with a worsening of hypertransaminasemia. The marked elevation of serum aminotransferase levels, observed in these children during the treatment, could be related to the rapid elimination of infected hepatocytes by IFNinduced activation of the cytolytic response (44). Three of these children, who were LKM-l-positive, had persistently undetectable HCV RNA throughout the treatment period. It is noteworthy that the coexistence of persistently undetectable HCV RNA and ALT worsening occurred only in LKM-l-positive patients. This seems to point to a peculiar immunological response to viral components in LKM-l-positive patients during IFN therapy. The role of molecular mimicry between the HCV genome and the molecular target of LKM-1 autoantibodies could be investigated in order to explain this peculiar immunological response. A molecular mimicry between viral antigens and host proteins has been invoked as trigger of autoimmune diseases (45). As for viral hepatitis, it has been reported that HBV shares epitopes with the molecular target of ANA and SMA (46). According to previous observations, the low titer of autoantibodies, typically associated with autoimmune liver disease, was a common finding in children with CHC and did not seem to be due to IFN treatment (47). With the exception of ANA, autoantibody frequency did not change significantly during treatment. Although in our study the LKM-1 autoantibody was frequently associated with a significant biochemical deterioration (observed in three of four LKM-l-positive patients) during IFN treatment, the presence of LKM1 autoantibodies does not seem to affect the response to IFN, because one of the only two complete sustained responders was LKM-l-positive. In this study, patients considered responders both at the end of therapy and at the end of follow-up, as reported in Table 2, usually normalized ALT and cleared HCV-RNA within the first 3 weeks of treatment; only

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P. Pensati et al.

one patient, a complete sustained responder, had a late ALT normalization and undetectable HCV RNA, at the third month of therapy. While early ALT normalization is predictive of a biochemical response, early undetectable HCV RNA (occurring at the second administration of IFN in 44% of patients) is a frequent phenomenon also in nonresponders. A low baseline HCV RNA serum level was a predictive factor of response to IFN, similar to findings in adults (21-23). In fact, there was a significant difference in basal HCV RNA serum levels between patients who were responders at the end of treatment and nonresponders: none of the responders had basal viremia levels above I .5X IO6 Eq/ml and patients with viremia below 1X106 Eq/ml had a significantly higher probability of response. We are unable to comment on the role of genotype because the frequency of genotypes other than lb is very low in our patients. No complete sustained responder and only one biochemical responder had genotype 1b. The poor response in patients with genotype 1b does not seem to be related to basal viremia levels. In fact, patients with genotype 1b and low basal viremia did not have a favorable response to INF (Fig. 3). In summary, only a very low proportion of children affected by CHC and treated with (x-INF for 12 months have long-term viral suppression associated with biochemical quiescence. Low baseline viral load is an essential factor for IFN response. Because longterm ALT normalization tends to be associated with a better histological prognosis and because it was obtained in 20% of treated children, IFN therapy could be recommended in children with low basal viremia. In LKM-l-positive children, who in this study invariably had a virological response frequently associated with a worsening of hypertransaminasemia, IFN could be administered at a lower dosage, at least during the first months of therapy, to limit the detrimental effect of IFN on transaminases. Since it has been reported that in adult patients with CHC, therapy with IFN and ribavirin is more effective than IFN alone in inducing virologic and histologic improvement (48), this combination therapy should be considered in the future also in children with CHC.

References 1. Hoofnagle

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