Mercedes Ruiz-Moreno

Factors predictive of response to interferon therapy in children with chronic hepatitis B

BACKGROUND/AIMS The efficacy of interferon therapy in Caucasian children with chronic hepatitis B is similar to that in adults. However, little information is available about factors predicting response to this therapy in children. We have performed a univariate analysis to assess the strength of association between basal variables and response, and a multivariate analysis to determine the combination of basal variables which give the best prediction of response in terms of sensitivity and specificity. METHODS The basal parameters were studied in 50 children included in three different trials of interferon alpha therapy (360-700 MU/square meter of body surface, 2 or 3 times weekly for 12 to 24 weeks). RESULTS Of these, 18 (36%) were responders. In the univariate analysis, a higher histological activity (p < 0.05), a lower percentage of HBcAg-stained hepatocytes (p < 0.001), aspartate amino transferase and alanine aminotransferase levels (p < 0.05) and alanine aminotransferase peak prior to serum HBV-DNA clearance (p < 0.05), were associated to the response. In the multivariate analysis, the combinations of the percentage of HBcAg stained cells with alanine aminotransferase levels and with the histological activity index were the best variables for predicting response (sensitivity: 100% and specificity: 89% and 86%, respectively). CONCLUSIONS Factors predictive of response in children with chronic hepatitis B are similar to those found in adults, and may help in identifying those children with a better chance of responding.

Hepatitis C virus infection in children

The prevalence of hepatitis C virus (HCV) infection is relatively low in childhood, with anti-HCV prevalence rates of 0.1-0.4% in the Western world. To date, blood transfusion has been the principal route of acquisition of HCV in children, but there is evidence that vertical transmission is overtaking it. The overall risk of vertical perinatal transmission of HCV is about 5%, although it increases with HIV co-infection and higher maternal viraemia. The mode of delivery and breastfeeding do not seem to affect the vertical transmission of HCV. Diagnosis of perinatal transmission relies on determination of ALT levels and the presence of HCV after the second month, while maternal anti-HCV antibodies may persist until 18 months of life. After infancy, a variable percentage of perinatally infected children are anti-HCV negative; thus, detection of HCV-RNA is necessary for accurate diagnosis. The natural history of HCV in childhood is not well understood and the outcome depends on host and viral factors. The rate of progression to chronicity is about 60-80% in both post-transfusion and vertically acquired HCV infection. Compared with adult patients, chronic hepatitis C in children is characterized by both low ALT levels and low viral load, as well as by the mildest histological and immunohistochemical forms of chronic hepatitis. The prognosis is usually worse in multitransfused, thalassaemic children and those who have had cancer. Experience of treatment of chronic hepatitis C in children is limited, with about 40% having a sustained response to the interferon therapy. It is necessary to perform long-term follow-up and multicentre treatment studies to improve knowledge of the natural history of HCV in children, as well as that of the efficacy of anti-viral therapy in childhood.

Distribution of the predominant hepatitis B virus precore variants in hepatitis B e antigen-positive children and their effect on treatment response

Little is known about the distribution of precore hepatitis B virus mutants and their influence on the outcome of interferon therapy in children with chronic hepatitis B. In this study, serum samples were analyzed from 60 children with chronic hepatitis B e antigen+ (HBeAg+) hepatitis. Fifty-two of these children underwent different interferon trials, and a second serum sample was taken from 25 of them at the end of therapy. Fifty-six of the original 60 children were simultaneously infected by wild-type and precore mutant hepatitis B virus variants. The remaining four children were infected by the wild type alone. In 50/56 of children with a mixed viral population, the wild-type variant comprised more than 50% of the total viremia. With respect to the influence of precore variants on the outcome of interferon treatment, the prevalence of mixed viral population was similar in responders and nonresponders (96 versus 88%, respectively). However, precore mutant variants were prevalent (>50% of the viral population) in 21% of the nonresponders, but in none of the responder children (p< 0.05). Viremia levels were significantly higher in nonresponder than in responder children (p < 0.05). No substantial changes in the prevalence of mutants were observed throughout the interferon therapy. In conclusion, mixed viral infection is found in a high percentage of children with chronic B HBeAg+ hepatitis. Response to interferon therapy does not seem to be related to the presence of hepatitis B virus precore mutants, but rather to the levels of viremia.

Hepatitis C Virus Genotypes in Serum and Liver of Children with Chronic Hepatitis C

Hepatitis C virus (HCV) 1a, 1b, 2a, and 2b subtypes have been studied in 30 serum samples and in 20 paired liver biopsies from children with chronic hepatitis C. One serum sample was negative to the four HCV subtypes studied. HCV 1b was detected alone in 53.3% (16/30) of the serum samples; HCV 1a alone in 23.3% (7/30) and a mixed infection in 20% (6/30). In liver samples, HCV mixed infection was detected in a significantly higher proportion (50%: p < 0.05) than in serum, whereas HCV 1b was detected alone in 35% (7/20) and HCV 1a alone in the remaining 15% (3/20) of liver samples. These results suggest that serum HCV genotyping may not reflect the viral population infecting the liver of a given patient. Additional studies should be performed concerning the pathobiology of hepatitis C virus with relation to the subtypes detected in liver.

Sequence variation of hepatitis B virus precore-core open reading frame isolated from serum and liver of children with chronic hepatitis B before and after interferon treatment

DNA and amino acid sequences of the hepatitis B virus (HBV) genome were studied in serum and liver samples taken from 12 children with chronic hepatitis B before and after interferon (IFN) therapy. The purpose was to discover whether the persistence of low levels of viral replication with normal alanine aminotransferases after the response to IFN treatment is due to the appearance of mutations in the sequence of HB core antigen T and B cell epitopes. The existence of mutants was studied by amplification of precore‐core region of the HBV genome by polymerase chain reaction (PCR) and direct sequencing of the PCR products. In addition to the wild type sequence, mutation 1896 in the precore region was detected in the baseline serum and liver samples of five children. No changes in the distribution were found in the final samples, except one case. In the core region, both the wild type sequence and amino acid substitutions were observed in the basal serum and/or liver samples of six patients and most of these remained detectable in the samples after treatment. Sixteen (67%) of 24 changes in the core amino acid sequences were found in the T‐ or B‐cell epitopes. The results suggest that viral persistence after response to IFN therapy in children is not due to the appearance of mutants in the HBV core T‐ and B‐cell epitopes and that the host immune response can control the viral replication. J. Med. Virol. 58:208–214, 1999.

EFFICACY OF INTERFERON ALPHA IN THE TREATMENT OF CHILDREN WITH CHRONIC HEPATITIS B

It has been reported (Lancet 1987; ii: 877-880) that recombinant interferon alpha (rIFN-α) is not useful in Chinese children with chronic hepatitis B. We carried out a controlled study in Caucasian children. We included 36 children (mean age 8.2 years) with an histologically proven chronic hepatitis. All of them were HBsAg, HBeAg and HBV-DNA positive for at least 1 year before entry into the study. All the children were anti-HD and anti-HIV-1 negative. They were randomly allocated to one of the following groups: group 1) 12 children received 10 MU/m2 b.s. of rIFN-α 2b (Intron A) subcutaneously thrice weekly for 6 months; group II) 12 children received 5 MU/m2 b.s. under the same schedule: group iii) 12 children, control without treatment. No basal differences between the three groups were observed with respect to age, sex, ALT, etc.The treatment was well tolerated and all children completed the therapy period. Side effects included flu-like syndrome, anorexia, myalgia, neutropenia and moderate thrombopenia.At the end of the treatment period, HBV-DNA became negative in 7 patients (59%) from Group I and 5 cases (42%) from Group II as compared to 2 (17%) from the control group (p < 0.05). This situation remained unchanged until the end of the trial (1 year). HBeAg was negative in 33% of the treated patients and only in 8% of the controls. The ALT was normal in 21% of the treated children and in 8% of the controls. Anti-IFN-α antibodies (as detected by ELISA) developed in 8 patients (4 with and 4 without HBV-DNA) and remained positive at the end of the treatment period.In summary, therapy with rIFN-α in children is well tolerated. A 6 months course of rIFN-α 2b induces a significant antiviral effect in caucasian children with chronic hepatitis B as compared with a control group.

PREDICTIVE FACTORS OF THE RESPONSE TO RECOMBINANT INTERFERON IN CHRONIC HEPATITIS B IN CHILDHOOD

The antiviral effect of recombinant interferon (rIFN) therapy of chronic hepatitis type B in children has been demonstrated. In order to determine the predictive factors of the response to rIFN, the basal features of responder and non-responder patients (age, sex, ALT levels, HBV-DNAp activity, time of HBsAg carriers, Knodells index and percentage of infected cells of HBcAg in the liver biopsy) have been evaluated. Twenty eight children (mean age 2-14 years) with viral replication markers (HBV-DNA positive) have been included. Eight children had received 10 MU of rIFN-a2C (Boehringer Ingelheim)/m2 body surface, I.M., twice a week during six months; 8 children were treated with 7.5 MU under the same conditions and 12 received 10 MU of rIFN-a2A (Roferon)/m2 body surface I.M., thrice weekly during 3 months. All of them had an histologically proven CAH. AT the end of the follow-up period (15 months) 8 children became HBV-DNA and HBeAg negative and were considered as rIFN-responders and 20 remained positive for these markers (non-responders).The responders patients had a significantly lower activity of HBV-DNAp (265.7± 323.6 vs 719.3± 480.3. p<0.05) and less percentage of HBcAg infected liver cells than the non-responders (14.17 ± 6.83 vs 58.28 ± 32.73 p<0.05). In addition, the ALT (210.5± 75.2 vs 128.8± 76.1, p<0.05) and the liver Knodells index of histological activity in the liver biopsies (12,25± 2.41 vs 7.64 ± 3.23 p<0.05) were higher in the responders than in the non-responders.In conclusion, the children who responded to rIFN therapy hod a more active liver disease (ALT, Knodells index). Furthermore these patients had a relatively low level of viral replication (HBV-DNAp, HBcAg in liver cells).

CHANGES IN THE LIVER HBV-DNA PATTERN EXPRESSION IN CHILDREN WITH HBV CHRONIC INFECTION

The antiviral effect of r-IFNa on HBV replication has been proven “in vivo”. However there is no information about the changes induced by the r-IFNa in the patterns of HBV-DNA, specially in children. The aim of this work was to study the effect of the r-IFNa over the liver HBV-DNA patterns in children with chronic hepatitis B.30 children with CAH histologically proved were included. All of them had HBeAg and serum HBV-DNA for at least 6 months prior to the beginning of the study. Two liver biopsies were obtained from all the patients, one just before the treatment and the second at the 15th month.HBV-DNA was tested in serum by dot blot and in liver by southern blot hybridization. In the first liver sample all children had replicative intermediates of the viral DNA and in one of them the HBV-DNA was also integrated in the host genome. In the second liver biopsy, in the children who did not respond to the therapy (HBV-DNA+ in serum) the replicative forms of the HBV-DNA remained in the liver and in 3 children inegrated HBV-DNA was detected simultaneously. Among the 9 patients who lost serum HBV-DNA at the end of the therapy, in 8 of them the viral DNA was undetectable in their liver, in the other child integrated and episomal forms non replicatives of the HBV-DNA were detected. None of the responder children lost the HBsAg in serum. To investigate this fact we looked for the presence of HBV-DNA in peripheral blood cells by dot-blot. None of these children showed viral DNA in these cells.In conclusion, HBV-DNA can be integrated in the host genome early in the natural history of the HBV- chronic infection. Our results suggest the antiviral effect of the r-IFNa of hepatic level. The persistence of HBsAg in the responder children is not maintained at expenses of HBV-DNA expression in mononuclear blood cells.