This study was planned as an open, one-armed, prospective trial. CDH patients who were admitted to the Department of Gastroenterohepatology, Istanbul Medical Faculty, during the period March 2000 to March 2001 were included in the trial. The study protocol was approved by the Local Ethics Committee, and informed consent was obtained from all patients.
All included patients (n = 19) were positive for HBV surface antigen (HBsAg), HDV total antibody (anti-HDV), and HDV RNA for a minimum of 6 months; demonstrated serum alanine aminotransferase (ALT) levels at least 1.3-fold higher than the upper limit of normal (normal range, 5 to 40 IU/liter) on two occasions during the preceding 3 months; had compensated liver disease with histologic evidence of chronic hepatitis; and were between 18 and 65 years old. The patients were excluded if they met any of the following criteria: presence of any other etiology of chronic liver disease or seropositivity for human immunodeficiency virus antibody (anti-HIV), associated serious medical illness, pregnancy or breastfeeding, hepatocellular carcinoma, decompensated liver disease, a white blood cell count lower than 3,000/mm3, or a platelet count lower than 50,000/mm3. The diagnosis of CDH was based on anti-HDV and HDV RNA positivities and histological findings of chronic hepatitis.
All patients were administered IFN-α2b (Intron-A; Schering-Plough, Cork City, Ireland) (10 million U, subcutaneously, three times weekly) and ribavirin (Rebetol; Schering-Plough) (1,000 to 1,200 mg/day, orally) for 24 months. All subjects were monitored for at least 6 months after therapy discontinuation. A liver biopsy specimen was taken prior to the beginning of therapy. All liver biopsy slides were assessed according to the scoring system of Knodell et al. (11
Every patient underwent a detailed physical examination during visits, twice in the first month, once a month in the following 5 months, and once every 2 months during the remaining period. Serial hematological and biochemical studies were done during visits. Serum HDV RNA was tested at the beginning of treatment, at months 12 and 18, at the end of treatment, and at the end of follow-up. Compliance with therapy and adverse events were assessed at each visit.
HBsAg, hepatitis B “e” antigen (HBeAg), antibody to hepatitis B “e” antigen (anti-HBe) (Sanofi Pasteur Diagnostics, Marnes la Coquette, France), and anti-HDV (Abbott Laboratories) were determined by immunoenzymatic assays. Antibody to hepatitis C virus (anti-HCV) was tested with third-generation UBI HCV EIA 4.0 kits (Organon Teknika, RM Boxtel, The Netherlands), and anti-HIV was tested with a Vironostika HIV Uni-Form II Plus O kit (Organon Teknika) based on the technique called sandwich enzyme immunoassay. HBV DNA was quantitated by a hybridization technique (Hybride capture system; Digene Corp., Gaithersburg, Md.). The lower detection limit of this assay was 5 pg/ml. HBV DNA and HbsAg were analyzed before and after treatment.
For the diagnostic HDV RNA PCR and genotyping PCR, HDV RNA was extracted by a method described by Boom et al. (2
). A 100-μl serum sample was used appropriately for the protocol. Then cDNA was purified by using 10 μl of extracted material, 100 ng of random primer (Roche; catalog no. 1034731), a 0.4 mM concentration of each deoxynucleoside triphosphate (dNTP), 20 U of RNase inhibitor (Sigma; R2520), 5 U of avian myeloblastosis virus reverse transcriptase (Roche; catalog no. 1 495 062), and 1× buffer (total volume, 25 μl of reverse transcription [RT] buffer). The cDNA mixture was incubated at 42°C for 1 h.
HDV RNA has been used for the diagnosis of patients by RT nested PCR (13
). In the first stage of PCR, 5 μl of cDNA was added to a PCR mixture consisting of 10 mM Tris HCl, 1.5 mM MgCl2
, 50 mM KCl, a 0.2 mM concentration of each dNTP, a 0.5 mM concentration of outer primers 5413 (5′-GCC CAG GTC GGA CCG CGA GGA GGT) and 8276 (5′-ACA AGG AGA GGC AGG ATC ACC GAC), and 2.5 U of Taq
DNA polymerase enzyme (Sigma; D1806) (total volume, 50 μl). The reaction mixture was incubated for 35 cycles of 94°C for 60 s, 55°C for 60 s, and 72°C for 60 s. After incubation at 72°C for 10 min, the process was finalized. The same PCR cycle conditions and reagent concentrations were used in the second PCR stage with inner primers 5414 (5′-GAG ATG CCA TGC CGA CCC GAA GAG) and 5415 (5′-GAA GGA AGG CCC TCG AGA ACA AGA) and 5 μl of the first PCR product. PCR results were analyzed by performing electrophoresis with 1.5% agarose gels. According to the results of the serial dilutions performed, it was determined that the cutoff value of the PCR assay was 1,000 copies/ml.
The genotype analyses were done by using RT-PCR and restriction fragment length polymorphism methods (10
). For the PCR stage, 10 μl of cDNA was added to the PCR mixture consisting of 10 mM Tris HCl, 1.5 mM MgCl2
, 50 mM KCl, a 0.2 mM concentration of each dNTP, a 0.5 mM concentration of primers 900s (5′-GCC GAC CCG AAG AGG AAA G) and 1280as (5′-GAA GGA AGG CCC TSG AGA ACA AGA), and 2.5 U of Taq
DNA polymerase enzyme (Sigma; D1806) (total volume, 50 μl). The reaction mixture was incubated at 94°C for 9 min and then for 40 cycles of 94°C for 45 s, 58°C for 30 s, and 72°C for 45 s. After incubation at 72°C for 10 min, the process was finalized. PCR results were analyzed by performing electrophoresis with 1.5% agarose gels. For HDV genotyping with restriction fragment length polymorphism analysis, digestion was performed with 5-μl samples containing 5 U of SmaI enzyme (Fermentas ER0662) and 1× buffer (total volume, 20 μl), and then the mixture was incubated overnight. Digested products were analyzed by electrophoresis in 2% agarose gels. The DNA patterns (227 and 178 bp) were determined to be related to genotype I in all samples.
The HBV genotype was also tested by single-strand conformation polymorphism analysis or by a research line probe assay (INNO-LiPA HBV genotyping; Innogenetics N.V., Cohent, Belgium), which contained probes specific for the six major genotypes (A to F).
A virological response was defined by the disappearance of HDV RNA in serum. Normalization of serum ALT level was accepted as proof of a biochemical response. These measures of both virological and biochemical responses were assessed at the end of treatment and at the end of the follow-up period. During the follow-up period, the reappearance of serum HDV RNA and an increase in serum ALT level from the normal range to more than 1.3 times the upper limit of normal were defined as indicating virological and biochemical relapses, respectively.
All patients who completed the treatment and follow-up period were evaluated. The results are expressed as means ± standard deviations. The Mann-Whitney test was used to analyze quantitative data, and the chi-square test was used to analyze qualitative data. A P value of <0.05 was considered to indicate statistical significance. All calculations were made using SPSS 10.0 for Windows.