Prevalence of hepatitis C virus—Associated mixed cryoglobulinemia after liver transplantation

Prevalence of Hepatitis C Virus-Associated Mixed Cryoglobulinemia After Liver Transplantation Gregory A. Abrahamian, *A. Benedict Cosimi, *Mary Lin Farrell, * David A. Schoenfeld, t Raymond T. Chung, * and Manuel Pascual* Hepatitis C virus (HCV) infection is associated with mixed cryoglobulinemia and membranoproliferative giomernlonephritis. After orthotopic liver transplantation (OLT), isolated cases of HCV-associated mixed cryoglobullnemia have been reported. We determined the prevalence and dinical characteristics of mixed cryoglobulinemia in HCV-infected liver transplant recipients at our institution. Between January 1991 and February 1998, a total of 191 OLTs were performed in 178 patients. Among these transplant recipients, 53 patients (29.8%) had positive serological test results for HCV infection by second-generation enzyme-linked immunosorbent assay. We studied 31 HCV-positive (HCV+) and 21 HCV-negative (HCV-) transplant recipients (control group). Renal and liver function studies were performed, and cryoglobulin, rheumatoid factor, C3, CA, and serum HCV RNA levels and genotype were determined. Results were compared using unpaired Student's t-test for continuous variables and Fisher's exact test for categorical variables. Baseline characteristics were similar between the groups. Six patients in the HCV+ group (19%) had mixed cryogiobnlins present at the time of evaluation compared with none in the HCV- group (P = .036). The only parameter associated with cryoglobulins in the HCV+ group was rheumatoid factor (P < .01). In 3 HCV+ patients with cryoglobl,lins, extrarenal signs of cryoglobnlinemia were present. Glomerulonephritis was found in 4 HCV+ patients. Two patients with purpura and cryoglobnlinemia had reduced clinical manifestations after antiviral therapy. In conclusion, mixed cryoglobulinemia was found in approximately 20% of the HCV+ liver transplant recipients. The presence of purpura or giomernlonephritis suggests HCV-associated mixed cryoglobulinemia, a dinical syndrome that may respond favorably to antiviral therapy. Copyright © 2000 by the American Association for the Study o f Liver Diseases

ince its initial report in 1990, the association between hepatitis C virus (HCV) infection and mixed cryoglobulinemia, with membranoproliferative glomerulonephritis, has been confirmed in several studies) -8 The description of an association between H C V infection and mixed cryoglobulinemia has been shown to be relevant for the treatment of cryoglobulinemia. In the nontransplant setting, treat-

S

ment with interferon-cx can reduce H C V viremia, cryoglobulin levels, proteinuria and stabilize renal function.5.6. 9 HCV-associated liver failure is a common indication for orthotopic liver transplantation (OLT).*° After OLT, H C V infection persists in most patients, and reinfection of the allograft occurs in 50% to 80% of the patients within the first year posttransplantation. 1°''2 We and others have reported cases of cryoglobulinemia or glomerular disease complicating OLT in HCV-infected transplant recipients. 13"17 However, the exact prevalence and clinical characteristics of these complications after OLT remain to be defined. The standard immunosuppression required in liver transplant recipients increases the level of viral replication, t2'ts'19 and this may promote the development of mixed cryoglobulinemia. Mixed cryoglobulinemia can present as a vasculitis with palpable purpura, arthralgias, and glomerular disease. 2°,21 When glomerular disease is present, nephritic or nephrotic syndrome with or without renal failure can occur. 15 The aim of the present study is to determine the prevalence of mixed cryoglobulinemia and/or glomerulonephritis in HCV-infected liver transplant recipients at our institution.

From the *Transplantation Unit and tCenter fir Biostatistics, Massachusetts GeneralHospitaland Harvard Medical School, Boston, MA. Presentedat the 25th Annual Meeting of the American Socie~ of TransplantSurgeons,May 19-21, 1999, Chicago,IL. Supported in part by the Helen and GeorgeBurr EndowedResearch and Educational Fund in Support of Transplantation (M.P.);grant no 022309 )%m the National Institutes of Health (R.T.C); and the Elsevier Research Initiative Award for the American Digestive Health Foundation (R. T.C.). Address reprint requests to Manuel Pascua£ MI~,, Renal and Transplantation Units, Box MZ 70, Massachusetts General Hospital Boston, MA 02114. Copyright © 2000 by the American Associationfir the Study of Liver Diseases 1527-6465/00/0602-003253.00/0

Liver Transplantatior~ Vol6, No 2 (March), 2000: pp 185-190

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Methods Between January 1991 and February 1998, a total of 191 OLTs were performed in 178 patients at the Massachusetts General Hospital (Boston, MA). From January 1991 to March 1994, patients were treated with cyclosporine, azathioprine, and corticosteroids. Since March 1994, tacrolimus has replaced cyclosporine as the calcineurin inhibitor for liver transplant recipients at our institution. The results and costs of OLT during that period and the immunosuppressive regimens used have recently been detailed. 22 Among these 178 transplant recipients, 53 patients (29.8%) were found to have a positive serological result for HCV infection pretransplantion by second-generation enzyme-linked immunosorbent assay (Ortho Diagnostic Systems, Raritan, NJ). Of these 53 HCV-infected transplant recipients, 18 patients died before the current study evaluation and 4 patients were not available for the study; thus, 31 patients were assessable. We studied the clinical, immunologic, and virological characteristics of these 31 HCV-infected (HCV+) liver transplant recipients. To compare the observations in HCV+ transplant recipients with those of a control group of patients with similar characteristics, we also studied 21 randomly selected liver transplant recipients without HCV infection (HCV-). The underlying diagnoses in the control group were alcoholic cirrhosis (n = 10), primary sclerosing cholangitis (n = 4), primary biliary cirrhosis (n = 3), alphalantitrypsin deficiency (n = 2), biliary atresia (n = 1), and cryptogenic cirrhosis (n = 1). Clinical information was reviewed for all patients from the day of transplantation, with no patients lost to follow-up. Informed consent was obtained from each patient. At the time of study evaluation, the following parameters were measured and recorded: serum creatinine level, 24 hour urine collection for determination ofproteinuria and glomerular filtration rate (GFR), and urinalysis for the detection of urinary casts and/or dysmorphic red blood cells. In addition, the following immunologic and virological data were obtained: cryoglobulin detection (cryocrit) and characterization (type ofcryoglobulin), complement values (C3, C4), rheumatoid factor, HCV RNA quantification by polymerase chain reaction (Roche Diagnostics, Somerville, NJ), and HCV genotype determination (Specialty Laboratories, Santa Monica, CA). All liver biopsy results of HCV-infected patients and, when available, renal pathological data, were reviewed. For the control group, a spot urine sample (with protein-creatinine ratio measurement) was used as a surrogate marker for 24 hour proteinuria, and GFR was estimated with the Cockcroft-Gault formula.

Statistical Analysis All results are expressed as mean -+ SD. Observations between groups were compared using the unpaired Student's t-test for continuous variables and Fisher's exact test for categorical variables. We analyzed the following clinical and

laboratory parameters: months from OLT to evaluation; type ofimmunosuppression (cyclosporine or tacrolimus); and C3, C4, rheumatoid factor, serum creatinine, proteinuria, GFR, and HCV viremia values. P values less than .05 were considered significant.

Results Patient Characteristics Patient characteristics of the H C V + group (n = 31) and H C V - control group (n = 21) are listed in Table 1. Age at OLT, time from O L T to evaluation, serum creatinine level just before OLT, and induction i m m u nosuppression (cylosporine or tacrolimus based) were similar between the 2 groups. Immunosuppression at the time of evaluation was cyclosporine based in 6 of 31 patients (19%) in the H C V + group and 6 of 21 patients (29%) in the H C V - group. The difference in immunosuppression between baseline evaluation and study evaluation reflected some crossover (from cylosporine to tacrolimus) that occurred in both groups because of recurrent episodes of allograft rejection. All patients in the H C V + group (n = 31) had detectable levels of viremia by polymerase chain reaction. At the time of evaluation, 22 of 31 patients (71%) had evidence of H C V graft hepatitis (biopsy-proven in 17 patients; chronically elevated liver function test results with high viremia titers in 5 patients). The mean viremia level was 3.01 + 0.64 million copies/mL in these 22 patients. The other 9 H C V + patients had no

Table 1. Baseline Patient Characteristics

Age at OLT (y) Women Creatinine at time of OLT (mg/dL) Time from OLT to evaluation (too) Induction immunosuppression Cyclosporine Tacrolimus Immunosuppression at evaluation Cydosporine Tacrolimus

HCV+ (n = 31)

HCV(n = 21)

47-+7 7/31 (23)

48 + 13 9/21 (43)

1.3 -+ 0.8

1.2 + 0.7

30 + 25

34 "4"29

10/31 (32) 21/31 (68)

7/21 (33) 14/21 (67)

6/31 (19) 25/31 (81)

6/21 (29) 15/21 (71)

NOTE. Values expressed as mean "4- SD or number of total (percent).

Cryoglobulinemia After Liver Transplantation

evidence of H C V graft hepatitis, determined by normal liver function test results at the time of evaluation. The mean viremia level in this group was 1.58 -+ 0.86 million copies/mL.

Immunologic Data Six patients in the H C V + group (19.4°/o) had mixed cryoglobulins present in serum at the time o f evaluation compared with none in the H C V - group (P = .036; Table 2). The mixed cryoglobulinemia was of type II (n = 3), type III (n = 2), and indeterminate (n = 1). The only parameter associated with the presence of mixed cryoglobulins in the H C V + group was rheumatoid factor (P < .01). There was also a trend for complement C4 (low C4 levels associated with cryoglobulins) that did not reach statistical significance (P = .07). Five of the 6 patients (83%) with mixed cryoglobulinemia also had evidence of H C V graft hepatitis (biopsy proven) compared with 17 of 25 patients (68%) with no cryoglobulins (P > .1). Similarly, there were no significant differences in viremia, H C V genotype, type of immunosuppresion, renal function (serum creatinine level or GFR), or complement levels between patients with (n = 6) and without cryoglobulins (n = 25). When comparing symptomatic (n = 3) to asymptomatic (n = 3) patients with cryoglobulins, we did not find significant differences in C3 or C4 level, viremia, or cryocrit. There was a trend, however, to greater titers of rheumatoid factor (300 - 155 v 67 + 49 IU/mL) in symptomatic patients with cryoglobulins (P = .07).

Table2. ImmunologicData at -time of Evaluation

Mixed cryoglobulinemia Low C3 (<80 mg/dL) Low C4 (<10 mg/dL) Elevated rheumatoid factory

HCV+ (n = 31)

HCV(n = 21)

6/31 (19)" 13/31 (42) 5/31 (16) 11/31 (36)

0/21 (0) 3/21 (14) 1/21 (5) 4/21 (19)

NOTE. Valuesexpressedas number of total (percent). *Three patients had type II; 2, type IIl; 1, indeterminate (insu~cient amount of cryoglobulin). tAll patients with cryoglobulinshad elevated rheumatoid factor levels.In the control group, the underlyingdiagnosis in patients with an elevated rheumatoid factor level was sclerosingcholangitis(n = 1), alcoholiccirrhosiswith idiopathic thrombocytopenic purpura (n = 1), and alcoholic cirrhosis(n = 2).

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Table3. Clinicaland Renal Dattaattune of Evaluation HCV+ (n = 31) Clinical manifestations Purpura/vasculitis* 3/31 (3 cryo +) GIomerulonephritist 4/31 (2 cryo +) Renal function studies Serum creatinine(mg/dL) 1.7 -+ 0.7 GFR (mL/min) 61 -+ 31 Urinary protein (g/24 h) 1.6 -+ 4.3 Proteinuria >1 g/24 h 7/31

HCV(n = 21) 0 0 1.5 +- 0.4 65 -+ 26 0.1 -+ 0.1 0/21

*Of the 3 patients with symptomaticcryoglobulins,vasculitis resolvedwith interferontherapy (n = 1) or spontaneously (n = I). In 1 patient, vasculitispersisteddespite interferon and ribavirintherapy. tThe 4 patients with glomerulonephritishad typicalurinary findings (red blood cell casts, dysmorphic red blood cells, and proteinuria) and, in 1 patient, the diagnosis was confirmed by biopsy (membranoproliferativeglomerulonephritis).

Clinical and Renal F u n c t i o n Studies at the Time o f Evaluation Clinical and renal data at the time of evaluation are listed in Table 3. Mean serum creatinine levels and GFRs were similar between the 2 groups. Proteinuria (protein > 1 g/24 h) was present in 7 of 31 H C V + patients (23%) compared with 0 of 21 H C V - patients (P = .02). The cause of proteinuria was glomerulonephritis (n = 4), cyclosporine toxicity with focal segmental glomerulosclerosis (n = 1), diabetic nephropathy (n = 1), and indeterminate (n = 1). In 3 of the 6 H C V + patients with circulating cryoglobulins, extrarenal signs of cryoglobulinemia (purpura and/or vasculitis) were present at the time of evaluation. Pretransplantation records showed that none of the patients with cryoglobulinemia had a history of purpura and/or vasculitis or glomerulonephritis before OLT, suggesting that cryoglobulinemia developed de novo after transplantation.

Response to Antiviral T h e r a p y Interferon-(x (3 million U, 3 times weekly) was used in 2 of the 3 patients with symptomatic cryoglobulinemia (both patients also had H C V graft hepatitis) with either a complete (Fig. 1) or partial resolution of symptoms. The patient with a partial response to interferon-or did not benefit from the subsequent addition o f ribavirin therapy to his regimen. In I

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Cryocrit 4 %

1%

1%

Purpura (vasculitis)

I

Illll 2.4

~z

4

2.2

3

2.0

.~

1.8

C.~

tinine

1.6

r~

|1

0

1

2

3

4

Months After Start of Interferon Figure 1. At the start of interferon therapy, cryocrit was 4% and there was active vasculitis and a nephritic sediment. After 4 months of therapy, HCV viremia decreased, with improvement in renal function and resolution of vasculitis. Cryocrit decreased to 1%.

patient, purpura resolved spontaneously without antiviral therapy. The cryocrit decreased in the 2 patients with complete resolution of vasculitis, whereas no change in cryocrit was noted in the patient with a partial response to interferon therapy.

Discussion

Although many recent reports have described the prevalence and relevance of HCV-associated mixed cryoglobulinemia in the nontransplantation setting, ]-7'2°'21 knowledge of this clinical syndrome after OLT remains preliminary. In the current study, we found that approximately 20% of the HCV-infected liver transplant recipients followed up in the outpatient clinic had detectable serum cryoglobulins. The observed prevalence in our series appears to be similar to that reported in the nontransplantation setting, i.e.,

not all patients with chronic HCV infection have HCV-associated mixed cryoglobulinemia.3,21 Larger studies will be needed to determine whether there is an increased prevalence of cryoglobulinemia after OLT. Another similarity with the nontransplantation setting is that only half the patients with detectable cryoglobulins in their serum had clinical signs of vasculitis, that is, pathological manifestations of cryoglobulinemia. 3'zl In our 31 HCV-infected liver transplant recipients, 3 of 6 patients with mixed cryoglobulins had active signs ofvasculitis and 4 patients (2 with cryoglobulins) had evidence of glomerulonephritis. It is interesting to note that proteinuria with or without nephrotic syndrome has been recently reported in HCV-infected liver transplant recipients. 13"w In contrast to the HCV+ patients, none of the HCVcontrols was found to have cryoglobulins or glomerulonephritis, a result consistent with the prevailing view

Cryoglobulinemia After Liver Transplantation

that mixed cryoglobulinemia is strongly associated with HCV infection. The only clinical or laboratory parameter associated with the presence of mixed cryoglobulinemia in our patients was rheumatoid factor, a finding consistent with the known rheumatoid factor activity of most mixed cryoglobulins (e.g., immunoglobulin M with antibody activity against the Fc portion of autologous immunoglobulin G). There was also a trend to low C4 levels, the second protein to be activated in the classical pathway of complement activation, in patients with cryoglobulins. Activation of the classical pathway of complement is seen in various immune complexmediated diseases and therefore, reduced levels of C4 after OLT may be of pathogenic significance and indicate the presence of mixed cryoglobulinemia. However, it is important to note that the principal site of C4 synthesis is the liver, and advanced liver disease caused by HCV graft hepatitis could also contribute to decreased C4 levels in serum. What are the implications of cryoglobulin detection in HCV-infected liver transplant recipients? Because generalized vasculitis involving the kidney can be a manifestation of cryoglobulinemia, the detection of symptomatic cryoglobulinemia, with or without recurrent graft hepatitis, may justify the initiation of antiviral therapy. Although the number of patients with symptomatic cryoglobulins in our series is small and does not allow us to draw firm conclusions, initiation of antiviral therapy was clearly associated with an improvement in clinical symptoms ofvasculitis, clearance ofviremia, and reduction in cryocrit in 1 patient (Fig. I). This result suggested that, as in the nontransplantation setting, 47'9 antiviral therapy may be beneficial for patients with mixed cryoglobulins after liver transplantation. In summary, we found that mixed cryoglobulinemia in a population of HCV+ liver transplant recipients is not uncommon, and its prevalence appears to be similar to that in nontransplantation patients with chronic HCV infection. The efficacy of interferon-ct and ribavirin remains to be studied in the subgroup of patients who present with symptomatic cryoglobulinemia.

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2. Agnello V, Chung RT, Kaplan LM. A role of hepatitis C infection in type II cryoglobulinemia. N Eng[ J Med 1992;327: 1490-1495. 3. Pascual M, Schifferli JA. Hepatitis C virus infection and glomerulonephritis. In: Andreucci VE, Fine LG (eds). International yearbook of nephrology. New York: Oxford University Press, 1996:20-26. 4. Johnson RJ, Gretch DR, Yamabe H, Hart J, Bacchi CE, Hartwell P, et al. Membranoproliferative glomerulonephritis ,associated with hepatitis C virus infection. N Engl J Med 1993;328:465-470. 5. Johnson RJ, Gretch DR, Couser WG, Alpers CE, Wilson J, Chung M, et al. Hepatitis C virus-associated glomerulonephritis: Effect of alpha-interferon therapy. Kidney Int 1994;46:17001704. 6. Stehman-Breen C, Alpers CE, Couser WG, Willson R, Johnson RJ. Hepatitis C virus associated membranous glomerulonephritis. Clin Nephrol 1995;44:141 - 147. 7. Johnson RJ, Willson R, Yamabe H, Couser W, Alpers CE, Wener MH, et al. Renal manifestations of hepatitis C virus infection. Kidney lnt 1994;46:1255-1263. 8. Sansonno D, Gesualdo L, Manno C, Schena FP, Dammaco E Hepatitis C virus-related proteins in kidney tissue from hepatitis C virus-infected patients with cryoglobulinemic membranoproliferative glomerulonephritis. Hepatology 1997;25:1237-1244. 9. Misiani R, Bellavita R Fenili D, Vicari O, Marchesi D. Interferon alpha-2a therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 1994;330:751-756. 10. Gane EJ, Portmann BC, Naoumov NV, Smith H, Underhill J, Donaldson PT, et al. Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med 1996;334:815820. 11. Chazouilleres O, Kim M, Combs C, Ferrell L, Bachetti R Roberts J, et al. Quantitation of hepatitis C virus RNA in liver transplant recipients. Gastroenterology 1994;106:994-999. 12. Gretch DR, Bacchi CE, Corey L, Delarosa C, Lesniewski R, Kowdley K, et al. Persistent hepatitis C virus infection after liver transplantation; Clinical and virological features. Hepatology 1995;22:1-9.

13. Burstein DM, Rodby RA. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. J Am Soc Nephrol 1993;4:1288-1293. 14. Rahamimov R, llan Y, Eid A, Shouval D, Tur-Kaspa R. Hepatitis C-associated cryoglobulinemia after liver transplantation. Transplantation 1995;60:1050-1055. 15. Pascual M, Thadhani R, Chung RT, Williams W, Mechan S, Rubin N, et al. Nephrotic syndrome after liver transplantation in a patient with hepatitis C virus-associated glomerulonephritis. Transplantation 1997;64:1073-1076. 16. Kendrick EA, McVicar JR Kowdley KV, Bronner M, Emond M, Alpers CE, et a[. Renal disease in hepatitis C-positive liver transplant recipients. Transplantation 1997;63:1287-1293. 17. Pham HP, Feray C, Samuel D, Gigou M, Azoulay D, Paradil V, et al. Effects of ribavirin on hepatitis C-associated nephrotic syndrome in four liver transplant recipients. Kidney Int 1998;54: 1311-1319. 18. Johnson MW, Washburn WK, Freeman RB, Fitzmaurice S, Dienstag J, Basgoz N, et al. Hepatitis C viral infection in liver transplantation. Arch Surg 1996; 131:284-291. 19. Araya V, Rakela J, Wright T. Hepatitis C after liver transplantation. Gastroenterology 1997;I 12:575-582.

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naemia in patients with chronic hepatitis C virus infection. Clin Exp Immunol 1996;104:25-31. 22. Gilbert JR, Pascual M, Schoenfeld DA, Rubin RH, Delmonico FL, Cosimi AB. Evolving trends in liver transplantation: An outcome and charge analysis. Transplantation 1999;67:246253.