Recurrence of hepatitis C virus genotype-4 infection following orthotopic liver transplantation: Natural history and predictors of outcome

BACKGROUND AND OBJECTIVES: There are few reports on hepatitis C virus genotype 4 (HCV-4) recurrences after orthotopic liver transplantation (OLT). Therefore, we undertook a study to determine the epidemiological, clinical and virological characteristics of patients with biopsy-proven recurrent HCV infection and analyzed the factors that influence recurrent disease severity. We also compared disease recurrence and outcomes between HCV-4 and other genotypes. PATIENTS AND METHODS: All patients who underwent OLT (locally or abroad) for HCV related hepatic cirrhosis from 1991 to 2006 and had recurrent HCV infection were identified. Clinical, laboratory and pathological data before and after OLT were collected and analyzed. RESULTS: Of 116 patients who underwent OLT for hepatitis C, 46 (39.7%) patients satisfied the criteria of recurrent hepatitis C. Twenty-nine (63%) patients were infected with HCV genotype 4. Mean (SD) for age was 54.9 (10.9) years. Nineteen of the HCV genotype 4 patients (65.5%) were males, 21 (72.4%) received deceased donor grafts, and 7 (24.1%) developed ≥1 acute rejection episodes. Pathologically, 7 (24.1%) and 4 (13.8%) patients had inflammation grade 3-4 and fibrosis stage 3-4, respectively. Follow-up biopsy in 9 (31%) HCV genotype 4 patients showed stable, worse and improved fibrosis stage in 5, 2 and 2 patients, respectively. Of the 7 patients in the recurrent HCV group who died, 6 were infected with genotype 4 and 4 of them died of HCV-related disease. CONCLUSION: This analysis suggests that HCV recurrence following OLT in HCV-4 patients is not significantly different from its recurrence for other genotypes.

Research Centre (KFSHRC), Riyadh, Saudi Arabia, from 1991 through the end of 2006 were identified through a computerized database. The study protocol was approved by the department and the local ethical and research committees. The patient charts, transplant information, and laboratory data were reviewed. Only patients with recurrent HCV infection were included in the analysis. Recurrent hepatitis C was defined as: 1) alanine aminotransferase (ALT) or aspartate ami-notransferase (AST) ≥ 2 times the upper limit of nor-mal; 2) positive HCV RNA polymerase chain reaction (PCR); and 3) liver biopsy consistent with recurrent hepatitis C, with no evidence of graft rejection. Patients were excluded from the study if they had liver biopsy evidence of acute rejection, biliary obstruction, ischemic hepatitis, or any other viral infections. Demographic data, including age and gender of the recipient, type of OLT, donor age, presence and number of acute cellular rejection episodes, viral load pre-and post-transplant, HCV genotype and primary immunosuppressive ther-apy, were all collected. All patients were followed-up in the post-liver transplant clinic at least every 2 months based on their condition, with regular clinical, biochem-ical, imaging and virological examinations as appropri-ate, in addition to the pathological assessment.
Histopathological assessment was performed ac-cording to the METAVIR scoring system by a single liver histopathologist. 7 In our center, protocol liver bi-opsies are not practiced and the decision to perform liver biopsy is made if the serum ALT and/or AST levels are ≥ 2 times the upper limit of normal, or there is a consistent increase in direct bilirubin. The normal reference range of serum ALT and AST in our labora-tory is 10-45 IU/L. Data was collected initially on a specialized data collection form, imported into a Microsoft Excel work-sheet and finally transferred to the statistical package for social sciences (SPSS) version 11.0 (SPSS, Chicago, IL, USA) for analysis. Means of continuous variables were compared using the t test, non-parametric tests (Wilcoxon and Mann Whitney) or one-way analysis of variance (ANOVA), in addition to the post-hoc test (Tukey) as appropriate. The chi-square or Fisher exact tests were used to compare frequencies and proportions. Multivariate stepwise logistic regression analysis was performed to determine the independent predictors of fibrosis. A P value <.05 was considered statistically sig-nificant. Cumulative survival was calculated by the lifetable method (Kaplan-Meier analysis) from the date of transplantation until the last follow-up, death, or loss of follow-up. Survival of patients in different groups was compared by the log-rank test.

RESULTS
Of the 116 patients who received transplants due to hepatitis C and were followed in our center, 46 satisfied all three inclusion criteria for having recurrent HCV in-fection post-OLT. Of these 46, 33 (71.7%) were males and the mean (SD) for age was 54.5 (11.2)  At the time of graft biopsy, 14 (30.4%) patients were treated with either tacrolimus or cyclosporine alone, while the remaining 32 (69.6%) received a combination of drugs, mostly either cyclosporine and mycopheno-late mofetil (MMF) or tacrolimus and MMF (Table  1). Twenty-six (56.5%) patients were taking steroids at the time of biopsy, and only three (6.5%) received IV steroids for treatment of acute rejection episodes. None of our patients received OKT3 or any other anti-lym-phocyte preparation.
Patients who received OLT at KFSHRC (n=27) were similar to those who had OLT abroad (n=19) in demographic, clinical, biochemical, and virological vari-ables. No significant differences were found between patients who received grafts from living-related donors (n=16) compared with patients who received deceased-  donor grafts (n=30) in demographic, clinical, biochem-ical and virological variables except for HCV viral load at the time of biopsy, which was significantly higher in patients who received deceased donor grafts (P=.008). Twenty-nine patients (63.0%) began peginterferon alfa-2a (40 KD; Pegasys, F. Hoffmann-La Roche, Basel, Switzerland) 135-180 mcg per week subcutaneously and ribavirin (Copegus, F. Hoffmann-La Roche, 800-1200 mg per day orally) therapy for 48 weeks based on their initial histological scores (patients were treated if they had elevated ALT of more than 2 times the upper limit of normal and had grade 2 or more inflammation and stage 2 or more fibrosis on liver biopsy. Of these, 16 (55%) were negative for HCV RNA at the end of therapy and three of these 16 (18.8%) relapsed after the end of treatment, resulting in a sustained virological re-sponse of 44.8% (13 of 29). After a mean (SD) followup of 38.8 (40.8) months (range, 7.9 to 173 months), 4 patients (in addition to the 1 patient who had cirrhosis at the initial liver biopsy) developed cirrhosis; 7 (15.5%) eventually died of liver failure; 1 died of unrelated causes; and the remainder of the patients were still alive at the time of writing.
There was no significant difference between pa-tients with HCV-4 and those infected with other  (Table 3). More patients (65.6%) with HCV-4 had hypertension than patients infected with other geno-types (30.8%, P=.036). Seventeen (48%) patients received pegylated interferon and ribavirin therapy, nine of whom responded to therapy and two relapsed after the end of the treatment response, resulting in a sustained virological response of 41%. During 38.5 months of follow-up (range, 7.9-170.5 months), 9 patients had a repeat liver biopsy, 5 showed a stable fibrosis score, 2 showed worsening and 2 showed im-provement.
Eight of the 46 patients (17.4%) died during the follow-up. Of those, 6 were infected with HCV-4; of these, 4 died of severe recurrent cholestatic hepatitis C or chronic graft failure secondary to hepatitis C. No significant difference was found between genotype 4 and non-genotype-4 patients in terms of survival dur-ing the follow-up period (Figure 1a). Similarly, survival analyses using Kaplan-Meier curves and the log-rank test were similar in the patients who had DDLT and LDLT as well as among those who had OLT locally and abroad (Figure 1b and 1c).

DISCUSSION
Liver disease secondary to HCV infection is the most common indication for OLT worldwide, as well as in Saudi Arabia. 1,8 Whether receiving the transplant local-ly or abroad, the majority of Saudi patients who receive transplants due to hepatitis C are infected with HCV-4. 9,10 Although HCV genotype has not been shown to affect the natural history of chronic hepatitis C, 11 the effect of genotype following OLT is controversial. [12][13][14] There are only a few reports describing the natural his-tory of recurrent HCV-4 after OLT in patients infected with genotype 4, most of which are conflicting. [3][4][5][6] Gane et al reported on 14 patients with recurrent HCV-4 post-OLT and found that about 50% of these patients have progressive liver disease. 4 Similarly, an analysis of 182 patients transplanted for HCV in Australia and New Zealand (16 of whom had HCV-4) found that, among many factors studied in a univariate and multivariate analyses, genotype 4 was associated with an increased risk for re-transplantation and death. 5 In contrast, a study from another Australian center, including patients with HCV-4, showed that genotype 1b, not 4, was asso-ciated with higher recurrence rates after transplant. 6 In a more detailed study from the UK, 32 of 128 patients who underwent OLT for hepatitis C were infected with HCV-4. A statistically significant greater fibrosis pro-gression rate was observed in HCV-4 patients compared to non-genotype-4 ones, although their rates of survival were similar. 3 The authors attributed the difference be-tween these two groups to the significantly older donor age in the HCV-4 group and the ethnic background of these patients (predominantly Egyptian). They stated that: "donor livers that were suitable for transplantation but unsuitable or not needed for citizens of the United Kingdom (UK) were offered to non-UK citizens who formed a second waiting list." 3 This policy may have led to the selection of inferior grafts for HCV-4 patients, who are predominantly non-UK citizens, leading to inferior results in these patients. When reporting the histological findings, these investigators used the last available scored biopsy (showing significantly higher fi-brosis progression rates). However, they did not report the findings of the initial liver biopsy, which was done according to a day-six protocol. It would have been in-teresting to see if indeed the quality of grafts given to patients with HCV-4 was inferior to that of those given to other patients.
In the current study, we report the results of patients who have biopsy-proven recurrent hepatitis C infection and make comparisons between patients with HCV-4 and non-HCV-4 genotype. Contrary to previous re-ports, our study showed that there were no significant differences between these two groups in terms of epide-miological, clinical and histological factors and outcome. We found that in the initial liver biopsy, which was per-formed after a mean time from transplantation of more than 2 years, there were only four patients who had fi-brosis scores greater than stage 3. Two of these patients had worsening fibrosis on subsequent biopsies.
Although the majority of deaths in our study were in genotype-4 patients (6 of 8 deaths), there were only 4 hepatitis C virus-related deaths in the HCV-4-infected patients (13% mortality) after a mean (SD) of 14.7 (6.6) months of follow-up and we found no statistical differ-ence in survival between genotype-4 patients and pa-tients infected with other genotypes.The relatively good results seen in our HCV-4 patients (44.8% sustained viral response) may be explained by a number of factors. First, donor selection tends to be extremely conserva-tive in our center, as marginal livers are very frequently rejected. Second, our mean donor age (about 30 years), an important parameter in post-transplant outcome, especially in hepatitis C patients, is significantly lower than what has been described in other studies. 15,16 Third, only 6.8% of patients used IV steroids, and none used anti-lymphocyte preparations. Fourth, the HCV mean viral load at the time of transplant seems to be lower in our study compared to previously reported studies on the same genotype. Fifth, we based this analysis on the initial liver biopsy with the first elevation of liver en-- zymes, although the mean time from transplantation to initial biopsy was more than 650 days. Among many fac-tors included in our analyses, the only factor predictive of an advanced histological score was the HCV RNA level at the time of biopsy. Although some studies have shown a positive correlation between pre-transplant viral load and severity of HCV recurrence after trans-plantation, 17,18 others have not shown a consistent cor-relation between HCV viral load post-liver transplant and histological outcome. 12,13 Our data suggest that this correlation may be important in at least genotype-4 pa-tients. This is also supported by the fact that all patients who received antiviral therapy and responded to it had either stable or improved histological scores. Recently, treatment of hepatitis C post-OLT has been shown to improve survival in such patients, further suggesting a correlation between viral load and severity of disease. 19 The main strength of the present study is that it in-cluded patients who had transplantation locally in our center as well as patients who received transplantations abroad. In addition, it included patients who received deceased donor and living related grafts. It also included patients with genotype 4 of an ethnic background dif-ferent from the one described in most previous studies reporting on genotype-4 patients (Saudi and Egyptian patients). All these factors gave a wider variety of geno-type-4 patients, which allows study of this genotype in a more comprehensive fashion. Additionally, this study had a long follow-up time.
The outcome of recurrent HCV-4 appears to be not affected by the type of liver OLT (LDLT or DDLT). This may emphasize a greater role for host, viral, donor and therapeutic variables on disease severity. Although this is an issue that generated some debate recently, a recent study by Lawal et al 20 showed no difference in the cumulative incidence of histological recurrence of HCV post-OLT or in the survival between recipients of DDLT (n=32) and those with split liver transplants (n=17), which is consistent with our results. The same result was also shown by a retrospective study involv-ing 289 HCV patients, which concluded that LDLT does not increase HCV recurrence and progression. 21 However, both studies 20,21 came from areas where geno-type 1 infection predominates. Further studies are need-ed to assess the role of transplant type on the severity of recurrent HCV-4.
Our study suffers from all the weaknesses inher-ent to a retrospective analysis. In addition, because we have not been practicing protocol biopsy in our center, it was extremely hard to draw strong conclusions from the fibrosis progression results we have reported. In con-clusion, our study suggests that HCV recurrence post-OLT in genotype-4 patients does not seem significantly different from recurrence in other genotypes.