Genotype 3 hepatitis E virus (HEV) infection is an emerging disease in industrialized countries, which may lead to chronic hepatitis and cirrhosis in immunosuppressed patients (1–5). Scarce data regarding extrahepatic manifestations related to HEV infection have been published. Neurological symptoms and severe thrombocytopenia that occur during HEV infection have been reported (6, 7). Herein, we report on, for the first time, cases of HEV-related glomerulonephritis and described the outcome of kidney function in solid-organ transplant (SOT) patients with chronic HEV infection under antiviral therapy.
Kidney Parameters During HEV Infection
Compared with 4 months (median time, range 1–6 months) pre-HEV infection, there was no significant change in type, dose, or trough levels of immunosuppressive therapy (Table 1) at HEV diagnosis. However, there was a significant decrease in estimated glomerular filtration rate (eGFR) (Table 1). In kidney- and kidney-pancreas-transplant patients (n=36), GFR significantly decreased from 52.9±17.7 to 48.8±18.7 mL/min (P=0.04). When the three kidney-transplant patients with positive BK viremia were excluded from the analysis, there was still a statistically significant decrease in eGFR from 53.7±17.6 to 49.5±18.5 mL/min (P=0.04). In liver-transplant patients (n=15), eGFR slightly decreased from 63.3±17.2 to 56±18.05 mL/min; however, the difference was not statistically significant (P=0.09). In 15 of the 51 patients (29%), eGFR decreased by more than 20% compared with preinfection levels.
HEV RNA was looked for in the urine in 30 patients at acute phase and in all patients with chronic HEV infection and was found to be negative.
Before and at HEV infection, the proportion of patients with proteinuria more than 30 mg per day was 41% (21 patients). Proteinuria levels have significantly increased in four kidney-transplant patients at HEV diagnosis. Three of these four patients underwent a kidney biopsy (cases 1, 2, and 3). The fourth patients had undergone a kidney biopsy a few months before HEV infection, which had shown marked grade III interstitial fibrosis and tubular atrophy (IF/TA). Therefore, it was decided not to perform another kidney biopsy in this patient. In addition, de novo mild proteinuria occurred in one kidney-transplant patient who had undergone a kidney biopsy at chronic phase (case 4). Finally, a liver-transplant patient developed de novo elevated proteinuria level at chronic HEV phase that required a kidney biopsy (case 5).
Four other kidney-transplant patients underwent a kidney biopsy at acute phase because of decreased kidney function without proteinuria. Three had grade I IF/TA with no features of acute rejection or glomerular disease. The fourth patient, who was known to have BK virus nephropathy, only had features of this latter disease. During the chronic phase, four additional patients underwent a kidney biopsy for persistent decreased kidney function that developed during the previous acute phase without significant proteinuria. All patients had grade I IF/TA with no features of acute rejection or glomerular disease.
Clinical Cases Description
A 33-year-old man received a kidney allograft for malformative uropathy. After 38 months, he presented with an acute genotype 3f-HEV infection. No other viruses were detected. He was receiving tacrolimus, mycophenolate mofetil (MMF), and steroids. Blood pressure was 140/90 mm Hg. eGFR had decreased from 56 mL/min, at 2 months before admission, to 41 mL/min (Fig. 1A). Proteinuria had increased from 0.6 to 3.85 g per day. Albuminemia was 33 g/L. There were no microscopic hematuria, detectable cryoglobulinemia, or donor-specific anti-HLA alloantibodies (DSAs). Rheumatoid factor and C3 and C4 levels were within the normal ranges. HEV RNA was not detected in the urine. Histological analysis of a kidney-allograft biopsy showed glomerulitis and mild exudative proliferation. The glomerular basement membrane showed double contours. Immunofluorescence exhibited subendothelial deposits of IgM, IgG, and complement components. C4d and SV-40 staining were negative. On electron microscopy, subendothelial deposits were present (Fig. 1B–D).
Because of the presence of exudative proliferation, he received three methylprednisolone pulses (500 mg each), followed by 1 mg/kg of prednisone for 10 days, and then tapered to 5 mg per day. Angiotensin-converting enzyme (ACE) inhibitors were given. eGFR was improved and 24-hour proteinuria decreased slightly (1.5g/day); both remained stable thereafter. Six months postacute hepatitis, tacrolimus doses were significantly reduced. After 3 months, kidney function and proteinuria returned to within their baseline ranges when the patient was cleared of the virus. At 7 months after HEV clearance, eGFR was 55 mL/min and proteinuria was 0.5 g per day.
A 26-year-old man received a kidney transplant for IgA nephropathy. One year after transplantation, he developed a biopsy-proven relapse of IgA nephropathy. Thirteen years after transplantation, he presented with acute genotype 3f-HEV infection. He was receiving sirolimus, MMF, steroids, and ACE inhibitors. Blood pressure was 130/80 mm Hg. eGFR was unchanged at 35 mL/min. Proteinuria had increased from 2 to 4 g per day, without hypoalbuminemia. Type II cryoglobulinemia was detected (15 mg/L), but no DSAs. HEV RNA was not detected in the urine. The kidney-allograft histological analysis showed grade III IF/TA, proliferative endarteritis, and features of IgA nephropathy. Immunofluorescence exhibited mesangial deposits of IgA. Sirolimus therapy was maintained. Because of persisting HEV replication 6 months after HEV infection, he was given a 3-month course of ribavirin monotherapy at the daily dose of 600 mg leading to HEV clearance. Consequently, proteinuria decreased from 4 g per day at ribavirin initiation to 2 g per day at HEV clearance. Cryoglobulinemia had become undetectable. GFR had remained unchanged at 35 mL/min.
A 40-year-old man received a kidney transplant for IgA nephropathy. Two years after transplantation, he developed a biopsy-proven relapse of IgA nephropathy, which required three steroid pulses. Six years after transplantation, he presented with acute genotype 3f-HEV infection. He was receiving sirolimus, MMF, steroids, and ACE inhibitors. Blood pressure was 130/80 mm Hg. eGFR had decreased from 53 mL/min, at 2 months earlier, to 39 mL/min. Proteinuria had increased from 2 to 4 g per day, without hypoalbuminemia. Only type II cryoglobulinemia was detected (15 mg/L). No DSAs were detected. HEV RNA was not detected in the urine. Kidney-allograft histological analysis showed grade III IF/TA, proliferative endarteritis, and features of IgA nephropathy. Immunofluorescence exhibited mesangial deposits of IgA. Because of the elevated proteinuria, sirolimus was replaced by low-dose tacrolimus.
After 3 months, he presented with nephrotic syndrome (proteinuria: 4 g/d; albuminemia: 29 g/L). eGFR was 35 mL/min. On a repeated allograft biopsy, histological findings were unchanged. Tacrolimus doses were significantly reduced (target trough level: 2–3 ng/mL) leading to HEV clearance. However, proteinuria remained unchanged, and he evolved to end-stage renal disease 2 years after the HEV infection.
A 24-year-old man underwent a kidney transplant for Alport syndrome. Four years after transplantation, he was admitted with an acute genotype 3f-HEV infection. He was receiving sirolimus and steroids. eGFR had decreased from 83 mL/min, at 2 months before admission, to 80 mL/min. There was de novo mild microalbuminuria of 190 mg per day, without microscopic hematuria. ACE inhibitors were given. After 37 months, although serum HEV RNA was still positive (6 log copies/mL), he developed elevated proteinuria of 6 g per day, without hypoalbuminemia or microscopic hematuria. eGFR had decrease to 37 mL/min. Blood pressure was 140/90 mm Hg. Type III cryoglobulinemia was detected (15 mg/L). Rheumatoid factors and complement components were within the normal ranges. No DSAs were detected. HEV RNA was detected the urine. Kidney-allograft histological analysis showed grade II IF/TA. The glomerular basement membrane showed double contours. Immunofluorescence exhibited subendothelial deposits of IgG and C3 complement components. C4d and SV-40 stains were negative. On electron microscopy, subendothelial deposits were present. We suspected HEV-induced membranoproliferative glomerulonephritis (MPGN) and placed the patient on a course of rituximab (375 mg/m2/week for 4 weeks). Anti-HEV therapy was not given. Consequently, proteinuria decreased to 1 g per day and cryoglobulinemia became undetectable. However, kidney function remained impaired, and he evolved to end-stage renal disease 3 years after the suspected HEV-induced MPGN diagnosis.
A 58-year-old man underwent an orthotopic liver transplant for primary sclerosing cholangitis. He had a 20-year history of severe hypertension. Eight years after transplantation, he was admitted with an acute genotype 3c-HEV infection. He was receiving tacrolimus monotherapy. eGFR had decreased from 46.8 mL/min, at 2 months before admission, to 33.1 mL/min. There was no proteinuria and no microscopic hematuria. Six months later, although serum HEV RNA was still positive (5.45 log copies/mL), he developed elevated proteinuria (2.5 g/day), without hypoalbuminemia, and microscopic hematuria. eGFR was 35 mL/min. Blood pressure was 140/90 mm Hg. Autoantibodies, rheumatoid factor, complement, and cryoglobulinemia were negative. HEV RNA was not detected in the urine. Histological analysis of a kidney biopsy showed serious nephroangiosclerosis lesions, severe IA, and mononuclear glomerular and interstitial infiltrate. No specific glomerular disease pattern was observed. Immunofluorescence exhibited IgG and C3 along the tubular basement membrane. He was given ACE inhibitors.
Eighteen months after HEV infection, he was given a 3-month course of pegylated-interferon (IFN [135 μg/week]). HEV RNA became negative by month 3. At the beginning of the antiviral therapy, eGFR was 32.8 mL/min, proteinuria was 4.9 g per day, albuminemia was 32 g/L, and type III cryoglobulinemia was present (20 mg/L). Interestingly, under antiviral therapy, proteinuria declined progressively to 0.4 g per day by month 3 after the initiation of pegylated-IFN therapy, and to 0.2 g per day 3 months later without any change in ACE inhibitor dosage. Cryoglobulinemia had become undetectable. However, eGFR, which was 27 mL/min at the end of therapy, declined progressively thereafter, leading to dialysis 24 months later, that is, 11 years after the orthotopic liver transplantation.
Kidney Function in Patients Receiving Antiviral Therapy
Kidney function was assessed in 12 patients who had completed the 3-month course of antiviral therapy (nine patients who had received ribavirin monotherapy and three who were given pegylated IFN). The three remaining patients who were given antiviral therapy for chronic hepatitis were still on ribavirin monotherapy at last follow-up and were consequently excluded of this analysis. In all patients, serum HEV RNA was negative at the end of therapy. eGFR increased significantly, and overall proteinuria did not differ under therapy (Table 2). However, urine protein level, which was within the nephrotic range in one patient, decreased significantly under antiviral therapy (case 5). Cryoglobulinemia, which was detected in 70% of these patients (43 [14–60] mg/L) at the initiation of antiviral therapy, disappeared under antiviral therapy. Similarly, rheumatoid factors disappeared and the C3 complement component was slightly decreased under antiviral therapy. Immunosuppressant doses were unchanged during this time (data not shown).
The extrahepatic manifestations of HEV infection are not well known. Herein, we report that HEV infection may induce glomerular injuries.
We have previously reported kidney-function impairment in a kidney-transplant patient with acute HEV infection (8). In this study, as compared with 4 (range 1–6) months before the acute phase, we found a statistically but not clinically significant decreased eGFR (−5 mL/min) during the acute phase. Although we do not have a control group, the decreased eGFR seems to be related to HEV infection. Indeed it is unusual to observe a decrease in eGFR in stable transplant patient within a short period of time in the absence of an acute rejection episode, an infection episode, a modification in immunosuppressant type or dose, or a functional renal insufficiency. This was not the case in this study. The mechanism inducing the observed eGFR decrease at HEV infection is unknown and has to be confirmed. Kidney function was also assessed in 12 patients who had received an antiviral therapy that lead to HEV clearance. In these patients, eGFR, which was impaired at 6 months after HEV infection, remained impaired until antiviral therapy was started, despite decreases in calcineurin inhibitor doses. However, kidney function significantly improved after HEV clearance.
Hepatitis B virus and hepatitis C virus (HCV) are known to often induce glomerular disease that may occur in the presence and absence of cryoglobulinemia (9). In this study, histological analysis of one patient's kidney biopsy performed at acute phase revealed signs of exudative proliferation and features of MPGN (case 1). The temporal association between acute HEV infection and glomerular disease suggested that our patient had developed HEV-associated glomerulonephritis. Because of the presence of exudative proliferation, steroid pulses were given, resulting in slight improvement to the kidney parameters with persisting severe proteinuria. Interestingly, kidney function and proteinuria returned to within their baseline ranges when the patient was cleared of the virus. Two other patients, who have experienced an IgA nephropathy relapse before HEV infection (cases 2 and 3), had undergone a kidney biopsy at HEV diagnosis, for a significant increase in proteinuria level. In both cases, type II cryoglobulinemia was detected and only features of IgA nephropathy were observed. In these two cases, it is difficult to incriminate HEV infection in the IgA nephropathy relapse. However, we may wonder whether HEV may have triggered this relapse. Interestingly, in one of these two patients (case 2), proteinuria returned to its baseline value when he was cleared of the virus after ribavirin monotherapy. Furthermore, cryoglobulinemia had become undetectable. The patient, who developed de novo proteinuria during the acute phase, had undergone a kidney biopsy during the chronic phase for increased proteinuria and decreased eGFR (case 4), in which type III cryoglobulinemia was detected. Histological analysis of the kidney biopsy revealed features of HEV-induced MPGN.
Anti-HCV therapy is the first-line therapy for HCV-induced glomerulonephritis (10). However, because IFN-α is contraindicated in the setting of kidney transplantation because of a high risk of acute rejection (11), rituximab has been found to efficiently treat HCV-related glomerulonephritis in kidney-transplant patients (12). Recently, pegylated-IFN or ribavirin monotherapy has been found to effectively achieve HEV clearance in SOT patients (13–16). However, at the diagnosis of HEV-induced MPGN, the efficacy of anti-HEV therapy was not yet established. For this reason, our patient (case 4) was given rituximab therapy alone. This lead to a decreased proteinuria level and cryoglobulinemia became undetectable. Unfortunately, kidney function decreased progressively, leading to dialysis.
In addition, a liver-transplant patient with a long history of hypertension and who was chronically infected by HEV developed de novo proteinuria. Histopathological analysis did not reveal any specific pattern of kidney disease. This is not surprising as, in the setting of liver transplantation, histological kidney lesions are complex, multiple, and interrelated (17). Interestingly, this patient's proteinuria declined and disappeared when he was cleared of the virus after pegylated-IFN therapy.
The mechanism by which HEV may induce glomerular disease is not well established. We speculate that HEV- induced kidney disease is immune driven and is quite similar to that observed with HCV (9). Indeed, some patients presented with glomerular injuries, with or without cryoglobulinemia. However, cryoglobulinemia was present in all patients with high proteinuria level, except one. Unfortunately, we did not look for HEV RNA in the cryoprecipitate. In the subgroup who received antiviral therapy, cryoglobulinemia was detected in 70% of patients before therapy and became undetectable in all patients after HEV clearance. In some patients, proteinuria decreased after HEV clearance. We cannot exclude a direct nephropathogenic of HEV. However, we failed to detect HEV in our patients' urine.
In conclusion, HEV-associated glomerulonephritis seems to be an HEV-related extrahepatic manifestation. Further studies are required to confirm these observations.
MATERIALS AND METHODS
Between January 2004 and April 2010, 51 autochthonous cases of HEV infection in SOT patients were diagnosed in our institution (Table 3). HEV diagnosis was based on increased liver enzyme levels, positive serum HEV RNA, and increased anti-HEV IgG and anti-HEV IgM during posttransplant follow-up.
Chronic HEV infection was defined by the presence of persisting increased liver enzyme levels and positive serum HEV RNA for at least 6 months after the acute phase. Twenty-two of the 51 patients (43.2%) were cleared of the virus spontaneously within the first 6 months after infection (resolving group), whereas the 29 remaining patients (56.8%) evolved to chronic hepatitis (chronic group). Nine of the 29 chronic patients had a sustained virological response after immunosuppressant-dose reduction (18), that is, negative serum for HEV RNA 6 months after HEV clearance. Fifteen other patients were treated by either pegylated-IFN-α (n=5) or ribavirin monotherapy (n=10). The five remaining patients with chronic hepatitis either died (n=4) (decompensated cirrhosis [n=2], respiratory distress [n=1] or septic shock [n=1]) or were lost to follow-up (n=1).
Clinical and laboratory parameters were assessed at transplantation, during HEV infection, and during anti-HEV therapy. The eGFR was obtained by the Modified Diet Renal Disease Formula.
HEV Virological Methods
Anti-HEV IgM and IgG were detected using commercial EIAgen HEV IgG and EIAgen HEV IgM kits (Adaltis, Ingen, France). A real-time polymerase chain reaction method with a hydrolysis probe was used to detect HEV RNA (19). The detection limit for HEV RNA was 200 copies/mL. The genotype was determined by sequencing a 189-nucleotide fragment within the ORF2 gene (20).
Reported values represent means (±standard deviation) or medians (ranges). Proportions were compared by the Fisher's exact test. In each group, quantitative variables were compared by the nonparametric Friedman test for serial measurements and the Wilcoxon test. A P value less than 0.05 was considered to be statistically significant.
The authors thank Prof. P. Callard and Prof. G. Touchard for electronic microscopy analyses.
1. 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.
2. Haagsma EB, van den Berg AP, Porte RJ, et al.. Chronic hepatitis E virus
infection in liver transplant recipients. Liver Transpl 2008; 14: 547.
3. Dalton HR, Bendall RP, Keane FE, et al.. Persistent carriage of hepatitis E virus
in patients with HIV infection. N Engl J Med 2009; 361: 1025.
4. Kamar N, Garrouste C, Haagsma EB, et al.. Factors associated with chronic hepatitis in patients with hepatitis E virus
infection who have received solid organ transplants. Gastroenterology 2011; 140: 1481.
5. Ollier L, Tieulie N, Sanderson F, et al.. Chronic hepatitis after hepatitis e virus
infection in a patient with non-hodgkin lymphoma taking rituximab. Ann Intern Med 2009; 150: 430.
6. Fourquet E, Mansuy JM, Bureau C, et al.. Severe thrombocytopenia associated with acute autochthonous hepatitis E. J Clin Virol 2010; 48: 73.
7. Kamar N, Bendall RP, Peron JM, et al.. Hepatitis E virus
and neurologic disorders. Emerg Infect Dis 2011; 17: 173.
8. Kamar N, Mansuy JM, Esposito L, et al.. Acute hepatitis and renal function impairment related to infection by hepatitis E virus
in a renal allograft recipient. Am J Kidney Dis 2005; 45: 193.
9. Morales JM, Campistol JM, Dominguez-Gil B. Hepatitis C virus infection and kidney transplantation
. Semin Nephrol 2002; 22: 365.
10. Kamar N, Rostaing L, Alric L. Treatment of hepatitis C-virus-related glomerulonephritis
. Kidney Int 2006; 69: 436.
11. Rostaing L, Izopet J, Baron E, et al.. Treatment of chronic hepatitis C with recombinant interferon alpha in kidney transplant recipients. Transplantation
1995; 59: 1426.
12. Basse G, Ribes D, Kamar N, et al.. Rituximab therapy for de novo mixed cryoglobulinemia
in renal-transplant patients. Transplantation
2005; 80: 1560.
13. Kamar N, Rostaing L, Abravanel F, et al.. Pegylated interferon-alpha for treating chronic hepatitis E virus
infection after liver transplantation
. Clin Infect Dis 2010; 50: e30.
14. Haagsma EB, Riezebos-Brilman A, van den Berg AP, et al.. Treatment of chronic hepatitis E in liver transplant recipients with pegylated interferon alpha-2b. Liver Transpl 2010; 16: 474.
15. Kamar N, Rostaing L, Abravanel F, et al.. Ribavirin therapy inhibits viral replication in patients with chronic hepatitis E virus
infection. Gastroenterology 2010; 139: 1612.
16. Mallet V, Nicand E, Sultanik P, et al.. Brief communication: Case reports of ribavirin treatment for chronic hepatitis E. Ann Intern Med 2010; 153: 85.
17. Kamar N, Guilbeau-Frugier C, Servais A, et al.. Kidney histology and function in liver transplant patients. Nephrol Dial Transplant 2011; 26: 2355.
18. Kamar N, Abravanel F, Selves J, et al.. Influence of immunosuppressive therapy on the natural history of genotype 3 hepatitis-E virus infection after organ transplantation
2010; 89: 353.
19. Mansuy JM, Peron JM, Abravanel F, et al.. Hepatitis E in the south west of France in individuals who have never visited an endemic area. J Med Virol 2004; 74: 419.
20. Legrand-Abravanel F, Mansuy JM, Dubois M, et al.. Hepatitis E virus
genotype 3 diversity, France. Emerg Infect Dis 2009; 15: 110.