- Email: [email protected]
Polyomavirus BK Replication in Liver Transplant Candidates with Normal Renal Function
Polyomavirus BK Replication in Liver Transplant Candidates with Normal Renal Function A.P. Mitterhofer, F. Tinti, M. Mordenti, V. Pietropaolo, M. Colosimo, S. Ginanni Corradini, F. Chiarini, M. Rossi, G. Ferretti, F. Brunini, L. Poli, P.B. Berloco, and G. Taliani ABSTRACT Polyomavirus-associated nephropathy (PVAN) has a predilection for kidney rather than for other solid organ transplants such as the liver. Immunosuppression is widely recognized to be a major risk factor for PVAN development. Since end-stage liver disease (ESLD) patients are immunocompromised and immunosuppression is a major cause of BK virus reactivation, we sought to evaluate BK virus replication in patients listed for liver transplantation. From April to October 2010, we enrolled 20 patients listed for liver transplantation. BK virus load was measured by quantitative real-time polymerase chain reaction on plasma and urine samples. Viremia occurred in only 1 among 20 patients. We hypothesized that in ESLD patients, the low prevalence of BK virus infection may be related to the prevalent impairment of antibacterial immunity rather than to the viral-specific one. In BK virus reactivation, not only the immunodepressive state itself, but also the specific immunologic mechanisms involved may have a role. OLYOMAVIRUS BK (BKV) infection is ubiquitous in humans. Seroprevalence of specific BKV anti-capsid protein VP1 immunoglobulin G antibodies is 87% to 71% in adults from 20 to 59 years old.1 BKV establishes latency in epithelial cells of the urinary tract. Under conditions of immunosuppression, it can undergo reactivation, eventually leading to severe diseases, such as polyomavirus nephropathy (PVAN) in renal transplant and hemorrhagic cystitis in bone marrow recipients.2 Polymerase chain reaction (PCR) is a sensitive and specific BKV replication marker that has been adopted by KDIGO (Kidney Disease: Improving Global Outcomes) 2009 guidelines as the screening method for PVAN in kidney transplant recipients.3 Risk factors for PVAN are related to donor, graft, virus, and recipient characteristics. The importance of the graft as source of infection can be partially supported by PVAN predilection for kidney rather than for other solid organ transplants such as the liver.4 Furthermore, posttransplant immunosuppression is widely recognized to be a major risk factor for PVAN development.5 Although immunosuppressive therapy is administered to all solid organ transplant patients, induction therapy is more often used in renal transplantation and steroid is administered for longer periods of time.4 The primary role of reduced immune responses that control BKV replication has been confirmed by a recent study that demonstrated dendritic cell (DC) dysfunction in renal graft
P
recipients pre- and posttransplantation who developed PVAN.6 In other PVAN cases dysfunction has been observed of BK specific interferon-gamma (INF␥)-secreting cytotoxic T lymphocytes (CTL).5 End-stage liver disease (ESLD) patients awaiting transplantation (pre-OLT) are affected by chronic diseases such as cirrhosis that are associated with several immune system abnormalities.7,8 These patients present a high prevalence of bacterial infections that represent important causes of morbidity and mortality.9 We sought to study BKV viremia in pre-OLT patients Since ESLD patients are immunocompromised and immunosuppression is a major cause of BKV reactivation, we evaluated BKV replication among patients listed for liver transplantation. From the Department of Clinical Medicine, Nephrology and Dialysis Unit (A.P.M., F.B., F.T.); Department of Clinical Medicine, Gastroenterology (M.M., C.S.G.); Department of Science and Public Health (V.P., M.C., F.C.); Department of Infectious and Tropical Diseases (G.F., G.T.); and Department of General Surgery, Organ Transplant Unit “Paride Stefanini” (M.R., L.P., P.B.B.), 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
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.02.048
© 2011 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
1142
Transplantation Proceedings, 43, 1142–1144 (2011)
POLYOMAVIRUS BK REPLICATION
MATERIALS AND METHODS From April to October 2010, we enrolled 20 patients (15 men and 5 women) who were listed for liver transplantation. BK virus load was measured on plasma and urine samples by quantitative realtime PCR (Q-PCR). We recorded clinical data: age, gender, etiology of ESLD, presence of hepatitis C and B, and cytomegalovirus infection, diabetes, and Model for End-Stage Liver Disease (MELD) score. We excluded patients with transjugular intrahepatic portosystemic shunts, hepatocellular carcinoma, acute infections, and evidence of chronic renal failure. Real-time PCR to detect BKV replication in samples of plasma and urine has been previously described.10
Clinical Specimens Processing Using a 1-mm urine specimen incubated in lysis buffer and proteinase K (200 mg/mL), we performed 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. From blood samples collected in EDTA and centrifuged at 1376 g/s for 10 minutes, we 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 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, USA). 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, 72°C for 1 minute, at the end of which we read 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 the 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.
RESULTS
Patients characteristics are shown in Table 1. Viremia occurred in 1/20 patients (5%) and viruria in 1/20 (5%) patients listed for liver transplantation. Viremia and viruria were concordant in the same patient. DISCUSSION
BK virus primary infection is ubiquitous in humans, occurring during early childhood. In seropositive individuals with altered immunity, such as transplanted patients, viral replication can reactivate and progress, causing serious organ damage. Disruption of the balance between host immune control and BKV replication is generally viewed as a key element in viral pathogenesis. Immune responses control-
1143 Table 1. Characteristics of ESLD Patients Waiting for Liver Transplantation Variables
Age, y (mean ⫾ SD) Male, n (%) Etiology of ESLD, n (%) Alcoholic HCV HBV Hemocromatosis Primary biliary cirrhosis Cryptogenetic CMV IgG positivity HBV infection HCV infection MELD (mean ⫾ SD)
55 ⫾ 9 15 (75) 13 (65) 5 (25) 4 (20) 1 (5) 1 (5) 1 (5) 19 (95) 4 (20) 7 (35) 16 ⫾ 5
CMV, cytomegalovirus; ESLD, end-stage liver disease; HBV, hepatitis B virus; HCV, hepatitis C virus; IgG, immunoglobulin G; MELD, Model for End-stage Liver Disease score; SD, standard deviation.
ling BKV infection involve specific antibody production, activation of antigen presenting cells, and emergence of specific INF␥-secreting cytotoxic T lymphocytes, the last elements considered to be the most important component for infection control.11 Many factors contribute to impaired immunity in ESLD patients. Bacterial translocation from the gut and endotoxemia are responsible for a chronic inflammatory state resulting in increased activation of T cells and monocytes accompanying increased apoptosis. T-regulatory lymphocytes proliferate in response to the immunologic hyperactivation, that intends to control the systemic inflammation.12 Furthermore, ESLD is characterized by dysfunction of Fc␥ macrophage receptors that are responsible for opsonization and clearance of microorganisms.13 Since ESLD patients are immunocompromised and BKV seroprevalence is high in the adult population, a high prevalence of BKV reactivation would be expected in these patients. Instead liver transplant patients, have been reported to show a low prevalence of BKV replication unlike kidney grafted subjects.14 To better understand BKV infection behavior, we evaluated BKV replication prevalence among an adult population of ESLD patients awaiting liver transplantation. Our results showed a low prevalence of BKV viremia; it was detectable in only one patient. Thus BKV infection may occur in ESLD patients, but its prevalence is low. We hypothesized that it could be related to the impaired antibacterial rather than viral-specific immunity. In contrast, renal transplant patients show a greater impairment of viral-specific immunity, as shown by altered DC and CTL functions, which may further explain the greater prevalence of BKV viremia in renal recipients. The main limitation of our study was the small sample size. This study offers a further contribution to our understanding of BKV behavior, allowing us to hypothesize that not only the immunodepressive state itself, but also the specific immunologic mechanisms involved may have a role in BKV reactivation.
1144
REFERENCES 1. 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 2. Jiang M, Abend JR, Tsai B, et al: Early events during BK virus entry and disassembly. J Virol 83:1350, 2009 3. 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 4. Puliyanda DP, Amet N, Dhawan A, et al: Isolated heart and liver transplant recipients are at low risk for polyomavirus BKV nephropathy. Clin Transplant 20:289, 2006 5. Egli A, Köhli S, Dickenmann M, et al: Inhibition of polyomavirus BK-specific T-cell responses by immunuppressive drugs. Transplantation 88:1161, 2009 6. 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 7. Navasa M, Rimola A, Rodes J: Bacterial infection in liver disease. Semin Liver Dis 17:323, 1997
MITTERHOFER, TINTI, MORDENTI ET AL 8. Christou L, Pappas G, Falagas ME: Bacterial infection related morbidity and mortality in cirrosis. Am J Gastroenterol 2007, 102:1510 –17. 9. Yang YY, Lin H-C: Bacterial infections in patients with cirrhosis. J Chin Med Assoc 68:447, 2005 10. Mitterhofer AP, Pietropaolo V, Barile M, et al: Meaning of early polyomavorus-BK replication post kidney transplant. Transplant Proc 42:1142, 2010 11. 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 12. 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 13. 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 14. 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
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recipients pre- and posttransplantation who developed PVAN.6 In other PVAN cases dysfunction has been observed of BK specific interferon-gamma (INF␥)-secreting cytotoxic T lymphocytes (CTL).5 End-stage liver disease (ESLD) patients awaiting transplantation (pre-OLT) are affected by chronic diseases such as cirrhosis that are associated with several immune system abnormalities.7,8 These patients present a high prevalence of bacterial infections that represent important causes of morbidity and mortality.9 We sought to study BKV viremia in pre-OLT patients Since ESLD patients are immunocompromised and immunosuppression is a major cause of BKV reactivation, we evaluated BKV replication among patients listed for liver transplantation. From the Department of Clinical Medicine, Nephrology and Dialysis Unit (A.P.M., F.B., F.T.); Department of Clinical Medicine, Gastroenterology (M.M., C.S.G.); Department of Science and Public Health (V.P., M.C., F.C.); Department of Infectious and Tropical Diseases (G.F., G.T.); and Department of General Surgery, Organ Transplant Unit “Paride Stefanini” (M.R., L.P., P.B.B.), 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
0041-1345/11/$–see front matter doi:10.1016/j.transproceed.2011.02.048
© 2011 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710
1142
Transplantation Proceedings, 43, 1142–1144 (2011)
POLYOMAVIRUS BK REPLICATION
MATERIALS AND METHODS From April to October 2010, we enrolled 20 patients (15 men and 5 women) who were listed for liver transplantation. BK virus load was measured on plasma and urine samples by quantitative realtime PCR (Q-PCR). We recorded clinical data: age, gender, etiology of ESLD, presence of hepatitis C and B, and cytomegalovirus infection, diabetes, and Model for End-Stage Liver Disease (MELD) score. We excluded patients with transjugular intrahepatic portosystemic shunts, hepatocellular carcinoma, acute infections, and evidence of chronic renal failure. Real-time PCR to detect BKV replication in samples of plasma and urine has been previously described.10
Clinical Specimens Processing Using a 1-mm urine specimen incubated in lysis buffer and proteinase K (200 mg/mL), we performed 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. From blood samples collected in EDTA and centrifuged at 1376 g/s for 10 minutes, we 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 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, USA). 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, 72°C for 1 minute, at the end of which we read 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 the 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.
RESULTS
Patients characteristics are shown in Table 1. Viremia occurred in 1/20 patients (5%) and viruria in 1/20 (5%) patients listed for liver transplantation. Viremia and viruria were concordant in the same patient. DISCUSSION
BK virus primary infection is ubiquitous in humans, occurring during early childhood. In seropositive individuals with altered immunity, such as transplanted patients, viral replication can reactivate and progress, causing serious organ damage. Disruption of the balance between host immune control and BKV replication is generally viewed as a key element in viral pathogenesis. Immune responses control-
1143 Table 1. Characteristics of ESLD Patients Waiting for Liver Transplantation Variables
Age, y (mean ⫾ SD) Male, n (%) Etiology of ESLD, n (%) Alcoholic HCV HBV Hemocromatosis Primary biliary cirrhosis Cryptogenetic CMV IgG positivity HBV infection HCV infection MELD (mean ⫾ SD)
55 ⫾ 9 15 (75) 13 (65) 5 (25) 4 (20) 1 (5) 1 (5) 1 (5) 19 (95) 4 (20) 7 (35) 16 ⫾ 5
CMV, cytomegalovirus; ESLD, end-stage liver disease; HBV, hepatitis B virus; HCV, hepatitis C virus; IgG, immunoglobulin G; MELD, Model for End-stage Liver Disease score; SD, standard deviation.
ling BKV infection involve specific antibody production, activation of antigen presenting cells, and emergence of specific INF␥-secreting cytotoxic T lymphocytes, the last elements considered to be the most important component for infection control.11 Many factors contribute to impaired immunity in ESLD patients. Bacterial translocation from the gut and endotoxemia are responsible for a chronic inflammatory state resulting in increased activation of T cells and monocytes accompanying increased apoptosis. T-regulatory lymphocytes proliferate in response to the immunologic hyperactivation, that intends to control the systemic inflammation.12 Furthermore, ESLD is characterized by dysfunction of Fc␥ macrophage receptors that are responsible for opsonization and clearance of microorganisms.13 Since ESLD patients are immunocompromised and BKV seroprevalence is high in the adult population, a high prevalence of BKV reactivation would be expected in these patients. Instead liver transplant patients, have been reported to show a low prevalence of BKV replication unlike kidney grafted subjects.14 To better understand BKV infection behavior, we evaluated BKV replication prevalence among an adult population of ESLD patients awaiting liver transplantation. Our results showed a low prevalence of BKV viremia; it was detectable in only one patient. Thus BKV infection may occur in ESLD patients, but its prevalence is low. We hypothesized that it could be related to the impaired antibacterial rather than viral-specific immunity. In contrast, renal transplant patients show a greater impairment of viral-specific immunity, as shown by altered DC and CTL functions, which may further explain the greater prevalence of BKV viremia in renal recipients. The main limitation of our study was the small sample size. This study offers a further contribution to our understanding of BKV behavior, allowing us to hypothesize that not only the immunodepressive state itself, but also the specific immunologic mechanisms involved may have a role in BKV reactivation.
1144
REFERENCES 1. 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 2. Jiang M, Abend JR, Tsai B, et al: Early events during BK virus entry and disassembly. J Virol 83:1350, 2009 3. 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 4. Puliyanda DP, Amet N, Dhawan A, et al: Isolated heart and liver transplant recipients are at low risk for polyomavirus BKV nephropathy. Clin Transplant 20:289, 2006 5. Egli A, Köhli S, Dickenmann M, et al: Inhibition of polyomavirus BK-specific T-cell responses by immunuppressive drugs. Transplantation 88:1161, 2009 6. 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 7. Navasa M, Rimola A, Rodes J: Bacterial infection in liver disease. Semin Liver Dis 17:323, 1997
MITTERHOFER, TINTI, MORDENTI ET AL 8. Christou L, Pappas G, Falagas ME: Bacterial infection related morbidity and mortality in cirrosis. Am J Gastroenterol 2007, 102:1510 –17. 9. Yang YY, Lin H-C: Bacterial infections in patients with cirrhosis. J Chin Med Assoc 68:447, 2005 10. Mitterhofer AP, Pietropaolo V, Barile M, et al: Meaning of early polyomavorus-BK replication post kidney transplant. Transplant Proc 42:1142, 2010 11. 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 12. 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 13. 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 14. 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