Hepatitis-E virus (HEV) infection is a classical cause of acute hepatitis in developing countries and an emerging cause of hepatitis in industrialized countries (1). Recently, HEV infection has been found to be responsible for chronic hepatitis in solid-organ transplant patients (2–4). Concomitantly, the first cases of HEV-related cirrhosis have been reported in kidney (5, 6), kidney-pancreas (6), and liver transplant patients (3). Scarce data have been published regarding the long-term evolution of HEV-induced liver fibrosis.
The evolution of HEV infection to chronicity seems to be, at least in part, related to intensity of the immunosuppressive therapy. Lymphocyte subset counts, mainly CD4+ lymphocyte counts, have been found to be significantly lower at HEV-infection diagnosis in patients who then evolve to chronicity compared with those who are cleared off the virus within 6 months (2). These data suggest that HEV clearance is mainly dependent on T cells. Herein, we report the outcome of HEV infection in 33 solid-organ transplant patients, the long-term evolution of liver fibrosis is described, plus the impact of immunosuppressive therapy on HEV infection.
PATIENTS AND METHODS
Between January 1, 2004 and October 31, 2008, among solid-organ transplant patients attending our outpatient and inpatient clinics, and who presented with unexplained acute elevation of liver-enzyme levels, we identified 33 patients with autochthonous acute HEV infection. Acute HEV infection was defined by the presence of increased liver enzymes levels and positive serum HEV RNA. Within the same period, 854 solid-organ transplantations have been performed in our center. The initial outcomes of the first 15 patients have been reported previously (2, 6). Six of these 33 patients, who had a follow-up of less than 6 months after HEV infection diagnosis, were not included in this study. Hence, we analyzed the outcome of the other 27 patients (17 kidney, 2 simultaneous kidney-pancreas, and 8 liver transplant patients). There were 24 men and three women ranging in age from 27 to 77 years (median, 49 years). All but two had received a graft from a deceased donor. The median number of transplantation was 1 (range, 1–3). The median time between transplantation and HEV diagnosis was 49.5 (range, 1–168) months. The other causes of hepatitis were ruled out (Table 1).
The causes of end-stage renal disease were glomerular disease (n=9), polycystic kidney disease (n=3), interstitial nephropathy (n=3), malformative uropathy (n=1), and thrombotic microangiopathy (n=1). Liver transplantation was needed because of hepatitis C virus (HCV)-induced cirrhosis (n=2), alcoholic-related cirrhosis (n=2), fulminant hepatitis B virus (HBV) (n=1), HBV-induced cirrhosis (n=1), Wilson's disease (n=1), and sclerosing cholangitis (n=1). The two kidney–pancreas transplant patients received transplants for diabetes mellitus. The two liver transplant patients who had received a transplant for HBV-related liver disease had negative HBs antigens and serum HBV DNA since transplantation, plus were receiving HBV-hyperimmune immunoglobulins and lamivudine prophylaxis. Serum HCV RNA was positive in only one patient. No patient received anti-HCV therapy.
Anti-HEV status was determined by the EIAgen HEV IgG kit, Adaltis (Ingen, France). HEV RNA from serum or stools was detected by a real-time polymerase chain reaction with Taqman detection, and an amplification of a 189-bp product located in the open reading frame 2 (ORF2) region (7, 8). The strain was sequenced and compared with reference HEV strains (GenBank) as previously reported (9). GenBank accession numbers: EU220992 to EU221003, FJ665420 to FJ665430, and FJ665979. The grade and stage of chronic hepatitis were assessed according to the Metavir classification (10).
Proportions were compared by the χ2 test or Fisher's exact test. Quantitative variables were compared with the nonparametric Mann-Whitney, Friedman, and Wilcoxon tests. A p value less than 0.05 was considered to be statistically significant. Risk factors for evolution to chronic hepatitis were defined using a multivariate, stepwise, logistical regression analysis that used initial inclusion criteria with a significance of P less than 0.05.
Among the 27 patients who were presented with acute HEV and considered for this study, 11 (40.75%) were cleared off the virus within 6 months of diagnosis (group of patients without chronic hepatitis). The remaining 16 patients (59.25%) developed chronic HEV-induced hepatitis, which was confirmed by the presence of persisting elevated liver-enzyme levels and positive serum HEV RNA at 6 months postdiagnosis (group of patients with chronic hepatitis).
Clinical and Biological Presentation at Diagnosis
The acute hepatitis episode was asymptomatic in 17 of the 27 patients. In these patients, HEV infection was searched for after liver-enzyme abnormalities were detected during routine biological examinations. The remaining 10 patients were presented with fatigue, diffuse arthralgias, and myalgias. One of them also had significant weight loss and was icteric. No abnormalities were detected during physical examination of any of other patients. All patients were born in France and none had traveled abroad during the year before diagnosis. Only, two patients reported having made contact with animals: chickens and rabbits for one patient, and birds for the second patient.
Biological and Histological Parameters and Immunosuppressive Therapies of Those Patients Without Chronic Hepatitis
The median follow-up in this group of 11 patients who were cleared off the virus within 6 months was 22 (range, 6–56) months.
These patients were spontaneously cleared off the virus at 1 month (n=7) or between months 1 and 3 (n=4). Thereafter, serum HEV RNA remained undetectable until the last follow-up.
At transplantation, anti-HEV IgG was assessed retrospectively in all patients for whom we had available stored sera (n=9) and was found to be negative in all cases. Within the 6 months before the acute phase, anti-HEV IgG was negative in all patients (n=11). At diagnosis, anti-HEV IgG was positive in two of the 11 patients (18%). Within the 6 months after acute hepatitis E diagnosis, five patients presented with HEV seroconversion (45.5%), whereas the remaining four patients did not develop anti-HEV IgG (36.5%).
Eight of the 11 serum-amplification products of HEV were sequenced and analyzed. We tried but failed to sequence the strains of the remaining three patients. Phylogenetic analysis revealed that seven of the strains belonged to genotype 3f and the eighth belonged to genotype 3c.
Liver Enzyme Levels
At diagnosis, liver-enzyme levels were significantly higher than those 3 (1–5) months earlier, but returned to the initial range within the 6 months after diagnosis, and then remained unchanged until the last follow-up (Fig. 1). Only one liver transplant patient had increased liver-enzyme levels at the last follow-up, and this was related to severe recurrence of HCV on the liver allograft.
Seven of the 11 patients underwent a liver biopsy at diagnosis. Activity and fibrosis scores were 2 (0–3) and 1 (0–1) Metavir units, respectively. The only liver transplant patient with positive serum HCV RNA, caused by recurrent HCV infection, underwent a second liver biopsy 10 months later. This showed an increased fibrosis score, whereas the activity score remained unchanged, that is, A1F2 versus A1F1. At this time, a repeat search for HEV RNA in the serum and stools was negative. Another patient underwent a second liver biopsy 22 months after diagnosis. This resulted in an improvement in activity score, A0F1 versus A2F1. None of the other patients who were cleared off the virus underwent a second liver biopsy.
Immunosuppressive drug type and levels were not changed significantly between diagnosis and 6 months later. Only one patient had his immunosuppressive therapy modified: this was a kidney transplant patient who presented with an HEV-related acute hepatitis at 1 month posttransplant. Tacrolimus and mycophenolate mofetil were stopped 1 month later because of a septic shock. A few days later, serum HEV RNA became undetectable. When mycophenolate mofetil was reintroduced, serum HEV RNA was again detected. At its discontinuation, few days later, HEV RNA became definitively undetectable.
Biological and Histological Parameters and Immunosuppressive Therapies for Patients With Chronic Hepatitis
The median follow-up in this group of 16 patients who evolved to chronic hepatitis was 27.5 (range, 7–96) months.
In those patients who evolved to chronic hepatitis, HEV RNA remained positive in the sera for at least 6 months after diagnosis. Twelve of the 16 (75%) patients were still viremic at 22 (range, 7–96) months after diagnosis, whereas the other four patients (25%) were cleared off the virus by 14, 16, 22, and 23 months after diagnosis. In these four patients, HEV RNA was still negative at 8, 12, 17, and 19 months after HEV clearance, and all these four were liver transplant patients.
At transplantation, anti-HEV IgG was assessed retrospectively in all patients for whom we had stored sera (n=15) and was found to be negative in all cases. At 6 months before the acute phase, anti-HEV IgG was found to be positive in one of the 16 patients. In this patient, HEV RNA was searched for retrospectively in many different samples and was found to be negative. At diagnosis, anti-HEV IgG was found to be positive in four patients (25%). Within the 6 months after acute HEV diagnosis, five other patients presented with HEV seroconversion (31.25%), whereas the seven remaining patients did not develop anti-HEV IgG (43.75%). At last follow-up, anti-HEV IgG was present in 13 of the 16 patients (81.25%), that is, in 10 of the 12 patients who were still viremic (83.3%) and in three of the four chronic patients who were cleared off the virus (75%) (p=ns; Table 2).
All 16 serum-amplification products of HEV were sequenced and analyzed. Phylogenetic analyses revealed that 13 of the strains belonged to genotype 3f, two belonged to genotype 3c, and one to genotype 3e. Among the four chronic patients who were cleared off the virus, three had HEV genotype 3f and the fourth had genotype 3e.
Liver Enzyme Levels
At diagnosis, by 6 months later, and at last follow-up, liver enzyme levels were significantly elevated compared with 4 (range, 2–8) months before acute HEV infection (Fig. 1). At last follow-up, liver enzyme levels were significantly greater in patients with chronic hepatitis. However, within the chronic group, both aspartate aminotransferase and alanine aminotransferase levels were significantly higher in patients who remained viremic compared with those who were cleared off the virus (Table 2).
Fourteen of the 16 patients with chronic hepatitis underwent a liver biopsy at diagnosis. Activity and fibrosis scores were 1 (range, 0–2) and 1 (range, 0–2) Metavir units, respectively.
In patients who remained viremic until last follow-up, 11, 9, 4, and 1 patients underwent, respectively, one, two, three, and four liver biopsies. The median (range) time between diagnosis and first, second, and third biopsies was 1 (0–9), 14 (10–38), and 26.5 (22–34) months, respectively. Between the first and last liver biopsy that was performed 22 (range, 10–96) months postdiagnosis, liver activity scores increased from 1 (range, 1–2) to 2 (range, 1–3) Metavir units (P=0.08). Conversely, liver-fibrosis scores increased significantly from 1 (range, 0–2) to 2 (range, 1–4) Metavir units (P=0.04). Three patients, that is, one kidney, one kidney–pancreas, and one liver transplant patient, had evolved HEV-related cirrhosis. The kidney and kidney-pancreas transplant patients were presented with decompensated cirrhosis and portal hypertension at 50 and 22 months postdiagnosis, respectively, and deceased from a hemorrhagic shock, as well as a septic shock and multiorgan failure.
In patients with chronic hepatitis who were later cleared off the virus, all patients underwent at least three liver biopsies, three patients underwent a fourth liver biopsy, and one patient underwent a fifth liver biopsy. The median (range) times between diagnosis and first, second, third, and forth liver biopsies were 0 (0–6), 11 (10–14), 20.5 (19–23), and 30 (27–31) months, respectively. The evolution of liver histology before and after HEV clearance is presented in Table 3. At diagnosis, liver activity and fibrosis scores did not differ significantly between patients who remained viremic and for those who were cleared off the virus. Conversely, on the last liver biopsy, activity scores were significantly higher in patients who remained viremic, whereas fibrosis scores were similar in both groups (Table 2).
The types of immunosuppressants used did not change significantly during follow-up (Table 4). Between diagnosis and 6 months later, median tacrolimus trough levels were significantly reduced (P=0.015). Conversely, other immunosuppressant levels and doses were unchanged. By the last follow-up, tacrolimus trough levels, mycophenolic acid, and steroid doses were significantly lower compared with HEV diagnosis time (Table 4). Comparison of immunosuppressive therapies given to those patients who remained viremic and to those who were cleared off the virus showed no significant differences at diagnosis and 6 months later. In contrast, tacrolimus trough levels and daily steroid doses were significantly reduced, and circulating positive CD3 and CD4 cells were significantly increased in patients who were cleared off the virus (Table 2). The number of patients who received an induction therapy was significantly lower in patients who were cleared off the virus. In one liver transplant patient, tacrolimus was stopped: this resulted in clearance of HEV. However, by 3 months later, the patient was presented with acute humoral rejection, which was treated with rituximab. Interestingly, HEV remained undetectable, despite rituximab therapy and the reintroduction of tacrolimus.
Differences Between Patients Without Chronic Hepatitis and Those Who Evolved to Chronic Hepatitis
Age, sex, the number of transplantation, the time since transplantation, the proportion of positive anticytomegalovirus IgG and anti-Epstein Barr virus IgG, and the proportion of patients with positive Epstein Barr virus DNAemia did not differ between both groups (data not shown). The proportion of patients receiving tacrolimus compared with cyclosporine A was significantly higher in the group of patients with chronic hepatitis (P=0.04). With respect to (1) the type and the daily dosage (mg/kg) of remaining immunosuppressive therapy, (2) the circulating levels of immunosuppressants, (3) total lymphocyte and lymphocyte-subset counts (CD2, CD3, CD4, CD8, and CD19), and (4) hemoglobin levels, and prothrombin index, no significant differences were observed between the two groups at diagnosis and at 6 months later (data not shown). The proportion of symptomatic patients at diagnosis was also similar in both groups. At diagnosis and by 6 months later, serum-creatinine levels were significantly increased in patients without chronic hepatitis. Conversely, platelets and white blood-cell counts were significantly lower in the chronic group (Table 5). The following significant factors identified by univariate analysis as associated with chronic HEV infection were included in a multivariate analysis model: the use of tacrolimus rather than cyclosporine A, serum creatinine level, and platelets count at diagnosis. No independent predictive factor for chronic HEV infection has been identified.
HEV infection was believed to only induce acute hepatitis, which is responsible in some cases for fulminant hepatitis that requires a liver transplant (1, 11). However, recently, cases of HEV-induced chronic hepatitis and cirrhosis have been reported in solid-organ transplant patients (2, 3, 5, 6). Herein, we present the results and outcomes for a large series of solid-organ transplant patients who developed acute hepatitis E. We found that (1) approximately 60% of patients evolved to chronic infection; (2) liver fibrosis increases rapidly in chronically infected patients; (3) interestingly, chronic patients in whom immunosuppressive drug levels were dramatically reduced could be cleared off the virus several months after the acute phase, and (4) the tacrolimus rather than cyclosporine A has been found to be more associated with HEV persistence.
In the Midi-Pyrénées area, that is, southwest France, the prevalence of anti-HEV IgG antibodies in blood donors from this area is found to be as high as 16.6% (12) and, excluding solid-organ transplant patients, 62 cases of acute hepatitis E have been identified (13). Two cases of prolonged HEV infection have been previously reported in hematological patients (8, 14). Recently, many cases of chronic HEV hepatitis have been reported in solid-organ transplant patients (2, 3, 5, 6, 15). Furthermore, five cases of HEV-related cirrhosis that occurred in two kidney, one kidney-pancreas, and two liver transplant patients have been reported (3, 5, 6). Herein, we identified 33 solid-organ transplant patients who presented with acute HEV infection, with other causes of hepatitis being ruled out. Among 27 of these 33 patients, who had a follow-up for longer than 6 months, 16 (59.25%) evolved to chronic hepatitis. In these 16 patients, liver-enzyme levels remained elevated, and HEV RNA was still detected in their serum at month 6. Serial liver biopsies showed a significant progression in liver activity and liver fibrosis. Three patients evolved to liver cirrhosis. The progression rate observed in this population seems to be more important than that shown in HCV-positive kidney transplant patients (16). Similar to previous reports, all patients had HEV genotype 3. Because genotype 3 HEV seems to be primarily an animal virus that occasionally crosses the species barrier, it has been speculated that genotype 3 HEV may be an opportunistic human pathogen causing infection and disease in humans only if the body resistance is low (17). No chronic hepatitis has been reported with HEV genotypes 1 or 2, which are primarily human pathogens.
In previous findings, we have shown that total lymphocyte and CD2, CD3, and CD4 lymphocyte-subset counts were significantly lower in patients who evolved to chronicity compared with those without chronic hepatitis, suggesting a higher immunosuppression in patients who evolved to chronic disease (2). In this study, the proportion of patients receiving tacrolimus rather than cyclosporine A has been found to be significantly higher in patients who evolved to chronic disease. This may be related to a higher immunosuppressive effect of tacrolimus compared with cyclosporine A. Indeed, after kidney, liver, or heart transplantation, the acute rejection rate has been found to be significantly lower in patients treated with tacrolimus compared with cyclosporine (18–20). Based on the previous finding (2), we significantly reduced tacrolimus trough levels, as well as mycophenolic acid and steroid doses in patients with chronic hepatitis. Interestingly, those who had a dramatic decrease in tacrolimus trough level were cleared off the virus. Indeed, in the four chronic patients who were cleared off the virus at 14, 16, 22, and 23 months after diagnosis, tacrolimus trough levels and daily steroid doses were significantly lower compared with those who remained viremic. This resulted in lower liver-enzyme levels and lower activity scores from liver biopsies. Peripheral blood CD3- and CD4-positive cell counts were significantly higher in these patients. All four patients were liver transplant patients, and only one had received an induction therapy with basiliximab. None had received antithymocyte globulins. Because the risk of acute rejection is lower after liver transplantation, immunosuppressive therapy can be lower than for kidney transplant patients. Comparing HEV with hepatitis G virus, it has been proposed that finding persistent HEV viremia in patients with elevated liver-enzyme levels does not necessarily imply that HEV infection leads to elevated alanine aminotransferase (17). We strongly disagree with this opinion because, as demonstrated in this study, the early or late clearance of HEV was associated with normalization of liver-enzyme levels, whereas those patients who remained viremic retained elevated liver-enzyme levels. In addition, activity scores observed in liver biopsies were significantly lower in patients who were cleared off the virus compared with those who remained viremic. Conversely, no significant differences were observed in fibrosis scores. This may be because patients who were cleared off the virus were liver transplant patients; therefore, the progression of liver fibrosis may be related to other causes than HEV, such as chronic allograft dysfunction.
The clearance of HEV after immunosuppressive drug-dose reduction and the higher circulating positive CD3 and CD4 cells counts in patients who were cleared off the virus suggest that HEV clearance relies mainly on T cells. In contrast to our previous report (2), CD2, CD3, and CD4 cell counts did not differ significantly between patients who evolved to chronic hepatitis and those who cleared off the virus. Lymphocyte subsets counts probably do not reflect the real immune status in this setting. Further immunologic studies are required to better understand the anti-HEV response, particularly in this immunosuppressed population, that is specific anti-HEV response. Hence, in the absence of an efficient antiviral therapy, reducing immunosuppressive therapy seems to be a therapeutic option. However, clinicians should exercise caution in this approach. One liver transplant patient in whom tacrolimus was interrupted, went on to develop an acute humoral rejection. Interestingly, after the reintroduction of tacrolimus and after rituximab therapy, HEV RNA remained undetectable, whereas anti-HEV IgG remained positive. It has been shown that antibodies to the HEV capsid can be protective against this disease (21). Passive immunoprophylaxis studies performed in cynomolgus monkeys confirms that the antibody to the hepatitis E virus capsid may prevent hepatitis E in humans (22). An HEV recombinant protein vaccine has been found to be effective in preventing hepatitis E infection (23).
Finally, the evolution to chronic hepatitis tended to be higher in liver transplant patients. This is probably related to the use of tacrolimus rather than cyclosporine A in this setting. In the other hand, interestingly, patients with chronic hepatitis who cleared off the virus after immunosuppressant dose reduction had received a liver transplant. This is because immunosuppressive therapy can be more reduced in liver transplant patients compared with kidney transplant patients because of the lower risk of acute rejection.
We acknowledge that statistical comparisons between patients with chronic hepatitis who remained viremic and those who cleared off the virus after immunosuppressant dose reduction should be considered with caution because of the small sample size.
In conclusion, approximately 60% of solid-organ transplant patients who present with HEV infection evolve to chronic disease. When possible, the reduction of immunosuppressive drugs targeting T cells should be considered as a first-line therapeutic option.
The authors thank Mrs. Martine Dubois for her technical assistance and Mr. Yohan Foucher for his statistical advices.
1. Dalton HR, Bendall R, Ijaz S, et al. Hepatitis E: An emerging infection in developed countries. Lancet Infect Dis
2008; 8: 698.
2. Kamar N, Selves J, Mansuy JM, et al. Hepatitis E virus
and chronic hepatitis
in organ-transplant recipients. N Engl J Med
2008; 358: 811.
3. Haagsma EB, van den Berg AP, Porte RJ, et al. Chronic hepatitis E virus
infection in liver transplant recipients. Liver Transpl
2008; 14: 547.
4. Gerolami R, Moal V, Picard C, et al. Hepatitis E virus
as an emerging cause of chronic liver disease in organ transplant recipients. J Hepatol
2009; 50: 622.
5. Gerolami R, Moal V, Colson P. Chronic hepatitis
E with cirrhosis in a kidney-transplant recipient. N Engl J Med
2008; 358: 859.
6. Kamar N, Mansuy JM, Cointault O, et al. Hepatitis E virus
-related cirrhosis in kidney- and kidney-pancreas-transplant recipients. Am J Transplant
2008; 8: 1744.
7. Mansuy JM, Peron JM, Bureau C, et al. Immunologically silent autochthonous acute hepatitis E virus
infection in France. J Clin Microbiol
2004; 42: 912.
8. Peron JM, Mansuy JM, Recher C, et al. Prolonged hepatitis E in an immunocompromised patient. J Gastroenterol Hepatol
2006; 21: 1223.
9. Legrand-Abravanel F, Mansuy JM, Dubois M, et al. Hepatitis E virus
genotype 3 diversity, France. Emerg Infect Dis
2009; 15: 110.
10. Bedossa P, Poynard T. An algorithm for the grading of activity in chronic hepatitis
C. The METAVIR Cooperative Study Group. Hepatology
1996; 24: 289.
11. Peron JM, Bureau C, Poirson H, et al. Fulminant liver failure from acute autochthonous hepatitis E in France: Description of seven patients with acute hepatitis E and encephalopathy. J Viral Hepat
2007; 14: 298.
12. Mansuy JM, Legrand-Abravanel F, Calot JP, et al. High prevalence of anti-hepatitis E virus
antibodies in blood donors from South West France. J Med Virol
2008; 80: 289.
13. Mansuy JM, Abravanel F, Miedouge M, et al. Acute hepatitis E in south-west France over a 5-year period. J Clin Virol
2009; 44: 74.
14. Tamura A, Shimizu YK, Tanaka T, et al. Persistent infection of hepatitis E virus
transmitted by blood transfusion in a patient with T-cell lymphoma. Hepatol Res
2007; 37: 113.
15. Gerolami R, Moal V, Picard C, et al. Hepatitis E virus
as an emerging cause of chronic liver disease in organ transplant recipients. J Hepatol
2009; 53: 622.
16. Kamar N, Rostaing L, Selves J, et al. Natural history
of hepatitis C virus-related liver fibrosis
after renal transplantation. Am J Transplant
2005; 5: 1704.
17. Aggarwal R. Hepatitis E: Does it cause chronic hepatitis
2008; 48: 1328.
18. Webster A, Woodroffe RC, Taylor RS, et al. Tacrolimus
versus cyclosporin as primary immunosuppression for kidney transplant recipients. Cochrane Database Syst Rev
19. McAlister VC, Haddad E, Renouf E, et al. Cyclosporin versus tacrolimus
as primary immunosuppressant after liver transplantation: A meta-analysis. Am J Transplant
2006; 6: 1578.
20. Ye F, Ying-Bin X, Yu-Guo W, et al. Tacrolimus
versus cyclosporine microemulsion for heart transplant recipients: A meta-analysis. J Heart Lung Transplant
2009; 28: 58.
21. Bryan JP, Tsarev SA, Iqbal M, et al. Epidemic hepatitis E in Pakistan: Patterns of serologic response and evidence that the antibody to hepatitis E virus
protects against disease. J Infect Dis
1994; 170: 517.
22. Tsarev SA, Tsareva TS, Emerson SU, et al. Successful passive and active immunization of cynomolgus monkeys against hepatits E. Proc Natl Acad Sci USA
1994; 91: 10198.
23. Shrestha MP, Scott RM, Joshi DM, et al. Safety and efficacy of a recombinant hepatitis E vaccine. N Engl J Med
2007; 356: 895.