Polyomavirus BK Infection Before Liver Transplantation in Patients With Chronic Kidney Disease

Polyomavirus BK Infection Before Liver Transplantation in Patients With Chronic Kidney Disease A.P. Mitterhofer, F. Tinti, I. Umbro, V. Pietropaolo, F. Fiacco, A. Bellizzi, E. Anzivino, S. Ginanni Corradini, L. Poli, M. Rossi, P.B. Berloco, G. Ferretti, F. Chiarini, and G. Taliani

ABSTRACT End-stage liver disease (ESLD) and chronic kidney disease (CKD) patients are both immunocompromised populations but polyomavirus BK (BKV) replication before liver transplantation is rare. We evaluated BKV prevalence among liver transplant recipients with renal dysfunction and the possible role of CKD as a risk factor for BKV replication in ESLD. From 2010 to 2011 we selected 31 ESLD patients awaiting liver transplantation to identify, the presence of CKD: No CKD (n ⫽ 22; 18 males) and CKD group (n ⫽ 9; 5 males). BKV infection was defined on the basis of viremia evaluated using quantitative real-time polymerase chain reactions. The prevalence of viremia among the No CKD group was 14% versus 56% in the CKD group (Fisher test; P ⫽ .027). We hypothesized that the presence of CKD may represent an additional condition of immunologic dysfunction regarding antiviral surveillances other than the antibacterial one that characterizes ESLD immunodysfunction, which could have promoted BKV replication. The specific immunologic mechanisms involved in pretransplantation diseases may have a role in BKV reactivation that could become responsible for nephropathy after transplantation. OLYOMAVIRUS BK (BKV) infection is asymptomatic in immunocompetent individuals, and can reactivate during immunosuppression resulting in viral replication in plasma and shedding in urine, eventually leading to severe diseases, such as polyomavirus nephropathy (PVAN).1 BKV infection is a significant cause of PVAN characterstics by dysfunction and graft loss in renal transplant recipients,2 but this phenomenon is rare in liver transplantation. In fact, BKV infection seems to have a predilection for the kidney rather than other solid organ transplants, such as the liver. Immunosuppression seems to be the major cause of BKV replication. End-stage liver disease (ESLD) patients awaiting orthotopic liver transplantation present several immune system abnormalities3,4 including a high prevalence of bacterial infections, which are important causes of morbidity and mortality.5 Chronic kidney disease (CKD) occurs among 1% of all patients with cirrhosis.6 Serum creatinine is a parameter of the Model for End-stage Liver Disease (MELD), which is the score used to prioritize liver transplantation.7 CKD patients are considered to be immunocompromised. Dendritic cell dysfunction has been documented in

P

kidney recipients who have developed PVAN, associated with cytotoxic T-lymphocyte dysfunction, supporting an antiviral immune dysfunction.8,9 In a previous study we evaluated the prevalence of BKV replication in liver transplant recipients with normal renal function10 and we hypothesized that the low prevalence detected in these patients could be related to the antibacterial rather than antiviral immunity impairment that characterizes liver immunodysfunction. The aim of our study From the Department of Clinical Medicine (A.P.M., F.T., I.U., F.F.), Nephrology and Dialysis Unit; Department of Science and Public Health (V.P., A.B., E.A., F.C.); Department of Clinical Medicine (S.G.C.), Gastroenterology Unit; Department of General Surgery (L.P., M.R., P.B.B.), Organ Transplant Unit “Paride Stefanini”; and Department of Infectious and Tropical Diseases (G.F., G.T.), 1st Faculty of Medicine and Surgery, Sapienza University of Rome, Rome, Italy. This study was supported by the Consorzio Interuniversitario per i Trapianti. Address reprint requests to Anna Paola Mitterhofer, Viale dell’Università 37, 00185 Rome, Italy. E-mail: annapaola.mitter@ uniromal.it

0041-1345/12/$–see front matter http://dx.doi.org/10.1016/j.transproceed.2012.06.052

© 2012 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710

1934

Transplantation Proceedings, 44, 1934 –1937 (2012)

POLYOMAVIRUS BK INFECTION

was to evaluate BKV prevalence among liver transplant recipients with renal dysfunction seeking to evaluate the possible role of CKD as a risk factor for BKV replication in ESLD. MATERIALS AND METHODS From April 2010 to January 2011, we enrolled 31 ESLD patients (23 males and 8 females) who were listed for liver transplantation. We recorded clinical data of age, gender, etiology of ESLD, presence of hepatitis C and B, cytomegalovirus infection, diabetes, hypertension, and MELD score. Renal function was evaluated based on serum creatinine, blood urea nitrogen, glomerular filtration rate (GFR) estimated with MDRD4 formula, and urinalysis. CKD was defined by a GFR ⬍60 mL/min/1.73 m2 for more than 3 months, according to the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines.11 BK virus load was measured on plasma and urine samples using quantitative real-time polymerase chain reaction (Q-PCR). BKV infection was defined according to the Kidney Disease Improving Global Outcomes (KDIGO) 2009 guidelines on the basis of viremia evaluated using Q-PCR12 considering copies of viral DNA per milliliter (c/mL) of samples ⬎1000. We excluded patients with transjugular intrahepatic portosystemic shunts, hepatocellular carcinoma, and acute infections.

1935 categorical variables. Nonparametric methods were used for inferential analysis. Independent variables were analyzed with the Mann-Whitney test. Chi-square analysis or Fisher exact test were used to compare categorical variables. All tests were performed using SPSS system 18.0 (SPSS Inc., Chicago, Ill, United States) with P ⬍ .05 considered significant.

RESULTS

We identified 2 groups of patients according to the presence of CKD: No CKD group (n ⫽ 22 [71%]; 18 males and 4 females) and CKD group (n ⫽ 9 [29%]; 5 males and 4 females). We did not observe any significant difference between the 2 groups in age, gender, etiology of ESLD, MELD score, presence of hepatitis C and B, cytomegalovirus infection, diabetes, and hypertension. Patient characteristics are shown in Table 1. The prevalence of viremia in the No CKD group was 14% (3/22) whereas it was 56% (5/9) in the CKD group (Fisher test; P ⫽ .027; Fig 1). The number of plasma copies of viral DNA/mL in the No CKD group was 10483 ⫾ 14152 versus the CKD group 18088 ⫾ 36722 (P ⫽ .094). In urine the prevalence of viral DNA positivity was 23% (5/22) in the No CKD group versus 12.5% (1/8) in the CKD group (P ⫽ 1.0) with 55506 ⫾ 114378 versus 1210 copies, respectively (P ⫽ .564).

Clinical Specimens Processing A 1-mm urine specimen incubated in lysis buffer and proteinase K (200 mg/mL) was subjected to DNA extraction using the DNeasy Tissue Kit (QIAGEN S.p.A., Milan, Italy) according to the manufacturer’s instructions. One microgram of purified DNA was used for Q-PCR. Blood samples collected in EDTA and centrifuged at 1376 g for 10 minutes were incubated (200 mL plasma) in lysis buffer and proteinase K (200 mg/mL). DNA extraction was performed using the QIAmp DNA Blood Kit (QIAGEN) according to the manufacturer’s instructions. One microgram of purified DNA was added to the Q-PCR. Urine and plasma samples were examined using quantitative assays to quantitate BKV DNA. The assay was performed using a 7300 real-time PCR System (AB Applied Biosystems, Foster City, Calif, United States). PCR amplifications were run in a reaction volume of 20 mL (optimized mix including forward and reverse primers and hydrolysis probes) containing 5 mL of total purified DNA (50 ng/mL). Thermal cycling was initiated with a first denaturation step of 10 minutes at 95°C, followed by 45 cycles of 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 1 minute, at the end of which we read the fluorescence. Amplification data were analyzed with software provided by the manufacturer. Standard curves to quantitate the viral genome were constructed using serial dilutions of a plasmid containing the target sequences (large T antigen). Plasmid concentrations ranged from 102 to 105 copies of target plasmid. All samples were tested in triplicate; the number of viral copies in each sample was calculated from a standard curve. The results were expressed as copies of viral DNA per milliliter (c/mL) of sample. We followed standard precautions to prevent contamination. In each run, we included nontemplate control lanes. The assay detected about 10 molecules of the target sequence in 5 mL DNA.

Statistical Analysis Data are presented as mean values ⫾ standard deviations (SD) for continuous variables, and as frequencies and percentages for

DISCUSSION

BKV infection is ubiquitous in the human population, occurring usually during early childhood, a seroprevalence of 65%–90% among children 6 –10 years old.13,14 BKV establishes a lifelong persistent infection in the kidney tubule and urinary tract epithelial cells.15 BKV infection is associated with a state of immunosuppression. Reactivation of human BKV entails plasma replication. In immunocompetent normal subjects, BKV infection evaluated by Q-PCR on plasma is in fact negative16,17 ESLD and CKD patients are both immunosuppressed. Table 1. Characteristics of 31 ESLD Patients Divided into 2 Groups on the Basis of Renal Function Variables

Age, y (mean ⫾ SD) Male, n (%) BMI (kg/m2) MELD (mean ⫾ SD) Etiology of ESLD, n (%) Viral Alcoholic Other HCV infection HBV infection CMV IgG positivity Diabetes Hypertension

No CKD Group (22 Patients)

CKD Group (9 Patients)

P

54 ⫾ 8 18 (81%) 27 ⫾ 5 16 ⫾ 5

58 ⫾ 6 5 (56%) 30 ⫾ 8 14 ⫾ 4

.81 .38 .37 .60

10 (45%) 7 (32%) 5 (23%) 7 (32%) 4 (18%) 20 (91%) 8 (36%) 5 (23%)

3 (33%) 2 (22%) 4 (44%) 3 (33%) 2 (22%) 9 (100%) 5 (56%) 4 (44%)

.61 .57 .39 .59 .13

Abbreviations: CKD group, ESLD patients with GFR ⬍60 mL/min/1.73 m2; No CKD group, ESLD patients with GFR ⬎60 mL/min/1.73 m2; BMI, body mass index; HCV, hepatitis C virus; HBV, hepatitis B virus; CMV, cytomegalovirus; IgG, immunoglobulin G; SD, standard deviation.

1936

Fig 1. Prevalence of viremia in No CKD group versus CKD group.

In our study, we observed a significantly high prevalence (56%) of BKV infection in plasma from the CKD group whereas it was not observed in the No CKD group (P ⫽ .027) whose prevalence was not different from normal subjects.16 Indeed, the mechanisms of immune dysfunction in the 2 groups of patients seem to be different. The immune response that controls BKV infection involves specific antibody production, activation of antigen-presenting cells (APC), and production of specific interferon (INF)␥secreting cytotoxic T lymphocytes.18 ESLD patients show an increased activation of T cells and monocytes accompanied by increased apoptosis as a result of the chronic inflammatory state secondary to bacterial translocation from the gut with endotoxemia.19 A prevalent dysfunction has been described among Fc␥ macrophage receptors that are responsible for the opsonization and clearance of microorganisms.20 CKD patients show prevalent disturbances of acquired immunity concerning mainly T lymphocytes and the APC. The interactions between T lymphocytes and APC are impaired predisposing to viral infections and inadequate responses to vaccinations. No significant difference has been shown in viruria prevalence and/or number of copies between the 2 groups, but viruria is not considered to be a specific marker of infection because BKV replication in tubular epithelial cells contributes less than 5% to the total urine BKV load.21 We hypothesized that the presence or renal dysfunction in our patients awaiting liver transplantation represented an additional condition of immunologic dysfunction regarding antiviral surveillance that could promote BKV replication in plasma. Moreover, renal transplant recipients show greater impairment of viral-specific immunity, as shown by altered dendritic cell and cytotoxic T-lymphocyte functions, which could explain the greater prevalence of PVAN observed among renal recipients. Therefore, although this study involves a small series, whose results should be confirmed in a larger number of patients, the presence of CKD seemed to be a risk factor for BK infection. In a previous study on 20 ESLD patients awaiting liver transplantation with normal renal function, only 1 patient showed a positive Q-PCR for BKV infection in plasma.10 The present study had confirmed a low prevalence of BKV

MITTERHOFER, TINTI, UMBRO ET AL

infection (3/22 patients) among ESLD patients with normal renal function. So we believe that, not only the immunocompromised state itself, but also the specific involved immunologic mechanisms may have roles in posttransplantation BKV reactivation. Liver transplant recipients show a lower incidence of PVAN than renal transplant recipients22 for unclear reasons. BKV infection from the donor’s kidney has a recognized role in renal versus other solid organ transplantation, but it does not completely explain the different prevalence of BKV infections in these 2 distinct transplant populations. The different prevalence of pretransplantation BKV infections could have a role in posttransplantation PVAN incidence so the main contribution of our study was to demonstrate different patterns of BK viremia in the 2 pretransplantation populations. REFERENCES 1. Ahsan N, Shah KV: Polyomaviruses and human diseases. Adv Exp Med Biol 577:1, 2006 2. Hirsch HH, Knowles W, Dickenmann M et al: Prospective study of polyomavirus type BK replication and nephropathy in renal-transplant recipients. N Engl J Med 347:488, 2002 3. Navasa M, Rimola A, Rodes J: Bacterial infection in liver disease. Semin Liver Dis 17:323, 1997 4. Christou L, Pappas G, Falagas ME: Bacterial infection related morbidity and mortality in cirrhosis. Am J Gastroenterol 102:1510, 2007 5. Yang YY, Lin HC: Bacterial infections in patients with cirrhosis. J Chin Med Assoc 68:447, 2005 6. Garcia-Tsao G, Parikh CR, Viola A: Acute kidney injury in cirrhosis. Hepatology 48:2064, 2008 7. Kamath PS, Kim WR, Advanced Liver Disease Study Group: The Model for End-stage Liver Disease (MELD). Hepatology 45:797, 2007 8. Egli A, Köhli S, Dickenmann M, et al: Inhibition of polyomavirus BK-specific T-cell responses by immunosuppressive drugs. Transplantation 88:1161, 2009 9. Womer KL, Huang Y, Herren H, et al: Dendritic cell deficiency associated with development of BK viremia and nephropathy in renal transplant recipients. Transplantation 89:115, 2010 10. Mitterhofer AP, Tinti F, Mordenti M, et al: Polyomavirus BK replication in liver transplant candidates with normal renal function. Transplant Proc 43:1142, 2011 11. National Kidney Foundation: K/DOQI Clinical Practice Guidelines for Chronic Kidney Disease: Evaluation, Classification and Stratification. Am J Kidney Dis 39(suppl 1):S1, 2002 12. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant 9(suppl 3):S1, 2009 13. Knowles WA: Discovery and epidemiology of the human polyomaviruses BK virus (BKV) and JC virus (JCV). Adv Exp Med Biol 577:19, 2006 14. Knowles WA: The epidemiology of BK virus and the occurrence of antigenic and genomic subtypes. In Khalili K, Stoner GL (eds): Human Polyomaviruses: Molecular and Clinical Prospectives. New York: Wiley-Liss, Inc.; 2001, p 527 15. Dörries K: Latent and persistent polyomavirus infection. In Khalili K, Stoner GL (eds): Human Polyomaviruses: Molecular and Clinical Perspectives. New York: Wiley-Liss, Inc.; 2001, p 197 16. Egli A, Infanti L, Dumoulin A, et al: Prevalence of polyomavirus BK and JC infection and replication in 400 healthy blood donors. J Infect Dis 199:837, 2009

POLYOMAVIRUS BK INFECTION 17. Hirsch HH, Randhawa P, AST Infectious Diseases Community of Practice. BK virus in solid organ transplant recipients. Am J Transplant 9(suppl 4):S136, 2009 18. Chen Y, Trofe J, Gordon J, et al: Interplay of cellular and humoral immune responses against BK virus in kidney transplant recipients with polyomavirus nephropathy. J Virol 80:3495, 2006 19. Márquez M, Fernández-Gutiérrez C, Montes-de-Oca M, et al: Chronic antigenic stimuli as a possible explanation for the immunodepression caused by liver cirrhosis. Clin Exp Immunol 158:219, 2009

1937 20. Ruiz P, Gomez F, Schreiber AD: Impaired function of macrophage Fc receptors and bacterial infection in alcoholic cirrhosis. N Engl J Med 331:1122, 1994 21. Funk GA, Gosert R, Comoli P, et al: Polyomavirus BK replication dynamics in vivo and in silico to predict cytopathology and viral clearance in kidney transplants. Am J Transplant 8:2368, 2008 22. Munoz P, Fogeda M, Bouza E, et al: Prevalence of BK virus replication among recipients of solid organ transplants. Clin Infect Dis 41:1720, 2005